ArticlePDF Available

From Traditional Breeding to Genome Editing for Boosting Productivity of the Ancient Grain Tef [Eragrostis tef (Zucc.) Trotter]

Authors:

Abstract and Figures

Tef (Eragrostis tef (Zucc.) Trotter) is a staple food crop for 70% of the Ethiopian population and is currently cultivated in several countries for grain and forage production. It is one of the most nutritious grains, and is also more resilient to marginal soil and climate conditions than major cereals such as maize, wheat and rice. However, tef is an extremely low-yielding crop, mainly due to lodging, which is when stalks fall on the ground irreversibly, and prolonged drought during the growing season. Climate change is triggering several biotic and abiotic stresses which are expected to cause severe food shortages in the foreseeable future. This has necessitated an alternative and robust approach in order to improve resilience to diverse types of stresses and increase crop yields. Traditional breeding has been extensively implemented to develop crop varieties with traits of interest, although the technique has several limitations. Currently, genome editing technologies are receiving increased interest among plant biologists as a means of improving key agronomic traits. In this review, the potential application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology in improving stress resilience in tef is discussed. Several putative abiotic stress-resilient genes of the related monocot plant species have been discussed and proposed as target genes for editing in tef through the CRISPR-Cas system. This is expected to improve stress resilience and boost productivity, thereby ensuring food and nutrition security in the region where it is needed the most.
Content may be subject to copyright.
plants
Article
From Traditional Breeding to Genome Editing
for Boosting Productivity of the Ancient Grain Tef
[Eragrostis tef (Zucc.) Trotter]
Muhammad Numan 1, Abdul Latif Khan 2, Sajjad Asaf 2, Mohammad Salehin 1, Getu Beyene 3,
Zerihun Tadele 4and Ayalew Ligaba-Osena 1,*


Citation: Numan, M.; Khan, A.L.;
Asaf, S.; Salehin, M.; Beyene, G.;
Tadele, Z.; Ligaba-Osena, A. From
Traditional Breeding to Genome
Editing for Boosting Productivity of
the Ancient Grain Tef [Eragrostis tef
(Zucc.) Trotter]. Plants 2021,10, 628.
https://doi.org/10.3390/
plants10040628
Academic Editor: Francesco Carimi
Received: 8 February 2021
Accepted: 22 March 2021
Published: 25 March 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1
Laboratory of Molecular Biology and Biotechnology, Department of Biology, University of North Carolina at
Greensboro, Greensboro, NC 27412, USA; m_numan@uncg.edu (M.N.); m_salehin@uncg.edu (M.S.)
2Natural and Medical Sciences Research Center, Biotechnology and OMICs Laboratory, University of Nizwa,
Nizwa 616, Oman; abdullatif@unizwa.edu.om (A.L.K.); sajjadasaf@unizwa.edu.om (S.A.)
3Donald Danforth Plant Science Center, St. Louis, MO 63132, USA; GDuguma@danforthcenter.org
4Institute of Plant Sciences, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland;
zerihun.tadele@ips.unibe.ch
*Correspondence: alosena@uncg.edu
Abstract:
Tef (Eragrostis tef (Zucc.) Trotter) is a staple food crop for 70% of the Ethiopian population
and is currently cultivated in several countries for grain and forage production. It is one of the most
nutritious grains, and is also more resilient to marginal soil and climate conditions than major
cereals such as maize, wheat and rice. However, tef is an extremely low-yielding crop, mainly due
to lodging, which is when stalks fall on the ground irreversibly, and prolonged drought during
the growing season. Climate change is triggering several biotic and abiotic stresses which are
expected to cause severe food shortages in the foreseeable future. This has necessitated an alternative
and robust approach in order to improve resilience to diverse types of stresses and increase crop
yields. Traditional breeding has been extensively implemented to develop crop varieties with traits
of interest, although the technique has several limitations. Currently, genome editing technologies
are receiving increased interest among plant biologists as a means of improving key agronomic traits.
In this review, the potential application of clustered regularly interspaced short palindromic repeats
(CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology in improving stress resilience
in tef is discussed. Several putative abiotic stress-resilient genes of the related monocot plant species
have been discussed and proposed as target genes for editing in tef through the CRISPR-Cas system.
This is expected to improve stress resilience and boost productivity, thereby ensuring food and
nutrition security in the region where it is needed the most.
Keywords: CRSIPR-Cas; drought tolerance; Eragrostis tef ; genome editing; stress resilience
1. Introduction
The world population is increasing at an alarming rate, demanding an increase in food
production. The Green Revolution of the 1960s has led to a substantial increase in major
cereal production, but that is unlikely to meet the urgent demand for higher food produc-
tion [
1
] under the current climate scenario. To meet world food demands, the production
of major crops alone is insufficient, as they are less suited to extreme climate and low-
input conditions [
2
]. There is an increasing interest in underutilized crops such as tef
(Eragrostis tef (Zucc.) Trotter); millets, including proso millet (Panicum miliaceum Mill.) and
finger millet (Eleusine coracana Gaertn.); and quinoa (Chenopodium quinoa Willd.), which
are more versatile due to their resilience to marginal growing conditions, and outstanding
nutritional values. Despite its valuable traits, the grain yield of tef is very low. In 2018,
the average yield of tef in Ethiopia was only 1.7 ton ha
1
as compared to maize (4 ton ha
1
)
and wheat (2.7 ton ha
1
) [
3
]. Tef is a cereal crop originating in the Horn of Africa, which is
Plants 2021,10, 628. https://doi.org/10.3390/plants10040628 https://www.mdpi.com/journal/plants
Plants 2021,10, 628 2 of 19
widely cultivated in Ethiopia and Eritrea. In Ethiopia, tef is a staple food for about 70% of
the population. The crop is annually cultivated on 2.9 million hectares of land, producing
about 4.5 million tons of grain [
4
]. Tef is tolerant to marginal soil and unfavorable climate
conditions, which makes it a potential crop for arid and semiarid areas as well as poorly
drained soils [
5
]. Tef is also one of the most nutrient-dense crops, containing high amounts
of macro- and micro-nutrients (primarily calcium and iron), amino acids and vitamins [
6
].
Tef cultivation in Ethiopia and around the globe has increased in recent years due to its
many health-related benefits. Since the absence of gluten epitopes has been confirmed
in tef by antibody assays [
7
], it has been recommended as an alternative diet for people
suffering from celiac disease, the immune reaction to consuming gluten containing foods
such as wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and rye (Secale cereal L.),
which affects 0.6–1.0 percent of the population globally [
8
,
9
]. In addition to the extensive
use of tef grain for human consumption, the straw of tef is more nutritious and palatable
as a livestock feed compared to the straw from cereals such as barley and wheat [
10
].
Moreover, tef straw is used as construction material because it serves as an organic binder
for mud used for plastering walls for local houses [
11
]. Various agronomic traits, such
as panicle architecture, tilling, grain size and plant height, have been targets for the im-
provement of tef yield. Grain yield is a highly complex trait which has several components,
including seed weight, form and size of panicles, florets per panicle and number of fertile
tillers [
12
,
13
]. Other important traits that determine grain yield include shoot biomass,
panicle weight and the number of tillers in a plant [
14
]. Furthermore, certain agronomic
traits such as shattering proneness, lodging tolerance, dry matter yield, leaf area, and plant
height directly or indirectly influence grain yield in crops [15,16].
The main factors causing yield loss in tef include susceptibility to lodging, weed
competition, drought, small grain size and soil acidity [
5
]. Although tef shows several
agronomic and nutritionally desirable traits, it is under tremendous pressure due to harsh
environmental stress conditions [
5
]. The crop is relatively resistant to diseases and insect
pests as compared to other cereal crops. Among abiotic stressors, tef yield is significantly
reduced by drought and soil acidity. Weed competition has broad about a range of effects
on the yield of tef in Ethiopia [
17
]. Many direct and indirect strategies of weed control
are employed by farmers [
18
]. Hand weeding and frequent tillage are the two commonly
used methods of weed control in tef production. Furthermore, weeds can be controlled
by herbicide application with proper management of spray times and frequency. How-
ever, the herbicides must be specific to broad-leaved weeds to avoid damaging tef plants.
Taken together, hand weeding, the use of herbicides and resistant tef varieties are viable
alternatives in order to overcome yield loss due to weeds. With proper weed control
methods, improved tef varieties such as Kora and Quncho have been shown to produce
higher yields [19].
Drought is a major abiotic stress which has significant effects on crop yield in most
African countries. Water scarcity has resulted in a fragile ecosystem in Africa’s arid and
semiarid regions. In sub-Saharan Africa, about 1.1 billion people live in drier environments;
however, this number is expected to double by 2050, and is expected to reach 2.5 billion
people [
20
]. Drought stress after planting [
21
] and during the flowering and grain filling
stage has serious effects on crop yields, and up to 60% of yield loss has been reported
in pearl millet at these stages [
21
,
22
]. In tef, drought has been reported to cause about 40%
yield loss [23].
The other major cause of low productivity in tef is lodging, which is the displacement
of the stalks from the vertical position due to wind and rain [
24
]. Lodging occurs frequently
before grain maturity, significantly reducing the grain yield [
25
]. Tef is primarily susceptible
to stem lodging [
26
,
27
]. Panicle length is also associated with lodging tolerance [
25
]. Semi-
dwarf varieties of tef are lodging-tolerant and produce higher yields than tall varieties [
28
].
Lodging limits the use of inputs such as N-fertilizers, exacerbating the susceptibility of
the plant to lodging [29].
Plants 2021,10, 628 3 of 19
To overcome the effects of the constrains mentioned above and to improve the tef
productivity, it is important to develop resistant and high-yield verities. There are several
approaches to increasing crop productivity as well as stress tolerance in crops. Among
these strategies, genome editing techniques have recently received increased attention.
Previous studies have suggested that the productivity of many cereal crops such as
maize [
30
,
31
], rice [
32
35
], wheat [
30
,
36
] and other monocots [
37
,
38
] have been improved
using the clustered regularly interspaced short palindromic repeats (CRISPR) system.
In rice (Oryza sativa L.), CRISPR-associated proteins (CRISPR-Cas) systems have been used
to improve tolerance to drought [
39
], cold [
40
] and salt stress [
41
,
42
], ultimately boosting
productivity [
39
]. In wheat, two efficient and simple CRISPR-Cas methods have been de-
veloped to improve productivity and stress resilience [
43
45
]. The CRISPR-Cas technology
used in these monocots is expected to be transferred to tef. Therefore, the aim of this review
is to highlight the potential of CRISPR-Cas-mediated gene-editing in trait improvement
in tef.
2. Mechanisms of Tolerance to Lodging and Environmental Constraints in Tef
2.1. Lodging Tolerance
Lodging is the process by which cereal shoots are displaced from an upright position
to a horizontal position [
46
]. Lodging is considered a complex phenomenon, influenced
by several factors, such as diseases, agronomic practice, crop history, soil type, landscape,
geography, rain and wind [
47
]. Stem lodging is the bending or breaking of stem internodes
(lower culm internodes), whereas root lodging is the failure of the root to maintain its in-
tegrity in the soil [
48
]. The application of fertilizers aggravates lodging, and hence the yield
potential of tef. Lodging stress can be reduced by controlling/decreasing plant height.
However, reducing plant height by inhibiting plant growth regulators or introducing
dwarfing genes could lead to crop yield reductions [
47
]; hence, researchers have suggested
targeting traits other than plant height to reduce yield loss due to lodging. A recent study
by Merchuk-Ovnat, et al. [
49
] suggested that early lodging is likely caused by a rapid
increase in inflorescence weight [
49
]. This group also observed variations among the tested
tef population in terms of lodging time and strength, with some populations possessing
the strength to hold the inflorescence in the grain filling season up to a certain point before
they were bent to the ground. Due to its weak stem, tef has high chance of succumbing to
lodging due to rain or wind [
50
]. Modification of the stem’s chemical composition, such as
its cellulose, lignin, structural carbohydrate and silica composition, is expected to increase
lodging-, disease-, and pest-resistance [
51
]. Silicon (Si) is a beneficial plant nutrient that has
been shown to increase tolerance to lodging, diseases and pests, as well as to abiotic stresses
such as drought, salinity, heavy metal stresses, and extreme temperature in various crops,
ultimately leading to increased grain yield [
52
56
]. We recently performed greenhouse
experiments to study whether tef benefits from Si application. Our findings revealed that
Si improves grain and biomass yield, stress resilience, and regulates the expression of
Si-transporter genes in tef [
57
]. However, conclusive evidence showing the mechanism of
silicon-induced stress resilience is lacking [58].
Although lodging is the main cause of low yield in tef [
59
], both physiological and
molecular aspects are understudied, and biotechnological, molecular and breeding tech-
niques [
47
] are not well developed to prevent lodging. A partnership formed by the ‘Tef
Improvement Project’ has recently developed semi-dwarf and lodging-tolerant tef varieties,
which are currently being disseminated in farmer’s fields in Ethiopia [60].
Lodging tolerance has been shown to be improved by modulating the biosynthesis
of plant growth regulators (PGRs). For example, the inhibition of gibberellic acid (GA)
has been shown to reduce plant height [
46
,
61
] and decrease lodging susceptibility. Shorter
internodes are associated with reduced plant height [
62
]. During the Green Revolution of
the 1960s and 1970s, inhibition or alteration of GA in rice and wheat was mainly targeted
for developing semi-dwarf varieties, which ultimately boosted the yield of these crops [
63
].
In tef, mutation in the
α
-Tubulin gene is associated with agronomically important traits
Plants 2021,10, 628 4 of 19
such as semi-dwarfism and lodging tolerance [
59
]. Blösch, et al. [
25
] have reported that
panicle angle contributes to lodging tolerance in tef. Jifar, et al. [
28
] also identified some
lodging tolerance genotypes (RIL-91,RIL-244 and RIL-11).
Genes associated with dwarfism in plants have been widely studied [
64
69
]. The two
prominent genes of the 1960s Green Revolution were the semi-dwarf (SD1) gene
in rice
[66,70,71]
and reduced height-1 (RHT-B1b and RHT-D1b) in wheat [
72
]. SD1 belongs
to the gibberellin biosynthetic pathway, whereas RHT is a GA response regulator and is
aDELLA protein family gene. DELLA proteins are important components of the signal
transduction pathway of GA, encoded by the wild-type allele of RHT-B1b and RHT-D1b [
73
].
In the Green Revolution wheat varieties, introduction of a stop codon in the N-terminus of
the two reduced height-1 (RHT-B1 and RHT-D1) loci was responsible for the semi-dwarf
and lodging tolerance traits [
72
]. In rice, the enzyme gibberellin 20-oxidase (GA20) encoded
by the SD1 gene is responsible for the biosynthesis of GA [
65
,
74
]. A frame shift mutation
due to a 383-bp deletion in the sd1 allele has been shown to greatly reduce the level of
GA20 oxidase [
66
]. Mutation of the sd1 and RHT homologs in tef could potentially lead
to lodging tolerance and significantly improve grain yield. Similarly, genetic loci (DW1,
DW2,DW3 and DW4) that control plant height across several environmental conditions
have been identified in sorghum. Recently, scientists have transferred these mutations
into a single sorghum line and managed to release a semi-dwarf commercial variety that
contains mutations in three loci (DW1,DW2 and DW4) [
75
,
76
]. This suggests that these
mutations could also be introduced into tef to develop semi-dwarf varieties with improved
stress tolerance and enhanced grain yield.
2.2. Drought Tolerance
Understanding the degree of stress tolerance in crop plants is important in devising
alternative strategies for improving yield and quality. Drought is one of the most important
abiotic stresses affecting plant growth and development. Plants have developed various
mechanisms of drought tolerance [
77
,
78
]. The mechanisms that have been reported in tef
include modifications of stomatal conductance, osmotic adjustment, development of a deep
rooting system and maintenance of cell membrane stability [
79
,
80
]. Development of
a deep root system and osmotic adjustment are major drought stress tolerance mechanisms
in many crops, including tef [
79
]. The association of plant height, root depth and thickness
to drought stress tolerance was previously reported in tef [
79
]. Recently, crosstalk between
plant height and drought tolerance was reported from a study on tef and other small cereals
where semi-dwarf plants were found to be drought-tolerant [
81
]. Osmotic adjustment is
also known to enable tef leaves to maintain leaf turgor pressure (LTP) [
79
,
82
] under extreme
drought conditions by retrieving and absorbing water even from dry soils. Modification of
root growth parameters in response to water scarcity is another strategy used to mitigate
drought stress [
83
,
84
]. For example, the increase in root length of cowpea, peanut and
soybean plants when exposed to drought enabled them to absorb deep soil water [
84
].
Similarly, developing deep-rooted tef plants with an extensive and broad root system is
a desirable trait to withstand drought stress [79].
2.3. Weed Competition and Herbicide Tolerance
Weed competition is another important plant trait in areas of low-input integrated
weed management systems [
85
]. The competitive ability of crops has been divided into
two broad categories; the first category is the crop’s ability to reduce competitor fitness,
whereas the second is the crop’s ability to resist yield losses and withstand its neighbor’s
competitive impact [
86
]. Different terms have been used for these aspects in the literature,
such as “tolerance ability” and “suppressive ability” [87,88].
In Ethiopia, smallholder farmers have adopted some cultural methods to mitigate
the impact of weed competition. Hand weeding and frequent tillage are common practices
used to control weeds in tef production [
17
]. Herbicides are not widely used, mainly due to
economic reasons and shortages of supplies. An alternative strategy in weed management
Plants 2021,10, 628 5 of 19
is the use of cultivars with competitive ability due to their sustainability [
88
,
89
]. However,
information on tef varieties with high weed competitive ability is limited as compared
to other cereals such as oats (Avena sativa L.), barley (Hordeum vulgare L.) and wheat
(Triticum aestivum L.) [
86
]. Tef varieties can be improved using genetic modification tools
such as the CRISPR system to improve weed tolerance and enhance productivity. Potential
genes for weed resistance and yield improvement can be overexpressed in tef or engineered
through the CRISPR-Cas system to minimize the impact of weed competition.
Herbicide-resistant varieties have been developed in crops such as soybean by tar-
geting key genes in amino acid synthesis or other functions. Among these genes, aceto-
lactate synthase (ALS) is involved in the synthesis of branched-chain amino acids such as
isoleucine, leucine and valine [
90
]. ALS is the target site for five non-competitive inhibitor
families—sulfonylaminocarbonyltriazolinones, pyrimidinylthiobenzoates, triazolopyrim-
idines, imidazolinones and sulfonylureas [
91
]. Plants engineered in the ALS gene are
resistant to non-selective herbicides, whereas all non-engineered plants, including weeds,
are sensitive to the non-selective herbicides. A similar principle was implemented to
develop glyphosate-resistant plants in which the EPSPS (5-enolpyruvylshikimate-3-phosphate
synthase) gene was targeted. The EPSPS gene is involved in the shikimate cycle [
92
]. Overex-
pression or knockout of the above-mentioned genes might contribute towards developing
tef plants with resistance to non-selective herbicides.
2.4. Panicle Architecture
Panicle architecture and grain size are important yield traits in cereal crops such as
rice, wheat and barley [
93
95
]. There is a direct relationship between agronomic traits
such as panicle number, number of spikelets in panicle, spikelet filling percentage, grain
size and number and crop yield [
96
]. For example, in rice, higher grain yield in a hybrid
variety is associated with the number of spikelets in a panicle [
96
,
97
]. In some crops, genes
that control panicle number and grain size have been identified and modified to increase
yield [
98
100
]. For example, OsSPL14 (squamosa promoter binding protein-like 14) gene
and microRNA “OsmiR397” promoted panicle branching and increased grain size in rice,
which ultimately lead to high grain yield [
99
,
101
]. In tef, homologs of these genes remain
to be identified and characterized to determine their role in increasing grain size and to
improve yield.
3. Status of Tef Improvement
3.1. Traditional Breeding: Past and Current Status of Tef Improvement
Scientific research on tef started in Ethiopia in 1950s [
102
]. Early breeding work fo-
cused on germplasm enhancement through collection, characterization, evaluation and
conservation, as well as genetic improvement in which pure lines were selected from
already existing germplasm [
11
,
103
] (Figure 1). Since flower opening characteristics were
revealed in tef in 1974, [
104
], hybridization was used as a means of tef improvement.
Molecular approaches in tef including marker development, genetic linkage maps, genetic
and molecular diversity analysis were initiated during 1995–1998 [
11
]. Further progress
was made during 1998–2003, including the initiation of interspecific hybridization,
in vitro
culture and mutagenesis in order to improve disease and lodging resistance. Over the last
two decades, there has been progress in the area of tef genetic architecture and genomics re-
search [
105
,
106
] (Figure 1). From a total of 42 improved tef varieties released by the National
Research Program in Ethiopia, 18 were developed using the hybridization technique [
107
].
Plants 2021,10, 628 6 of 19
Plants 2021, 10, x FOR PEER REVIEW 6 of 19
Figure 1. Improvement of tef varieties over the last 50 years. The improvement of tef started back
in 1970s with tissue culture techniques, followed by hybridization, the study of molecular diver-
sity, molecular marker analysis, the development of resistant varieties by interspecific hybridiza-
tion and mutation and the recently emerged clustered regularly interspaced short palindromic
repeats (CRISPR)-associated proteins (CRISPR-Cas) genome editing technique. Note: (The pictures
used in this figure were either taken in the author’s labs or drawn using ChemBioDraw software).
3.2. Molecular Marker Development
The application of molecular markers in tef improvement was initiated during 1995
1998 [11]. Molecular markers near target genes are utilized for marker-assisted selection
(MAS) or marker-assisted breeding (MAB) [108]. They enable the effective use of alleles
during the selection of phenotypes. The most commonly used markers are microsatellites
(simple sequence repeats; SSRs), amplified fragment length polymorphism (AFLPs) and
single nucleotide polymorphisms (SNPs) [108]. During the selection of molecular markers,
some important factors are considered, such as the quality and quantity of required DNA,
procedures for marker assays, the level of polymorphism and the cost of the marker [109].
In tef, the SSRs and expressed sequence tag (EST), restriction fragment length polymor-
phisms (RFLPs) and random amplified polymorphic DNA (RAPD) have been developed
[110,111]. Through SSR analysis, Abraha, et al. [112] identified and improved some im-
portant traits in tef, including grain yield, days to maturity, panicle length and plant
height. Similarly, variability in tef accessions was identified using AFLP markers, which
can be used in seed multiplication and breeding programs [113]. Application of these
Figure 1.
Improvement of tef varieties over the last 50 years. The improvement of tef started back
in 1970s with tissue culture techniques, followed by hybridization, the study of molecular diversity,
molecular marker analysis, the development of resistant varieties by interspecific hybridization
and mutation and the recently emerged clustered regularly interspaced short palindromic repeats
(CRISPR)-associated proteins (CRISPR-Cas) genome editing technique. Note: (The pictures used
in this figure were either taken in the author’s labs or drawn using ChemBioDraw software).
3.2. Molecular Marker Development
The application of molecular markers in tef improvement was initiated during 1995–
1998 [
11
]. Molecular markers near target genes are utilized for marker-assisted selection
(MAS) or marker-assisted breeding (MAB) [
108
]. They enable the effective use of alleles
during the selection of phenotypes. The most commonly used markers are microsatel-
lites (simple sequence repeats; SSRs), amplified fragment length polymorphism (AFLPs)
and single nucleotide polymorphisms (SNPs) [
108
]. During the selection of molecular
markers, some important factors are considered, such as the quality and quantity of re-
quired DNA, procedures for marker assays, the level of polymorphism and the cost of
the marker [
109
]. In tef, the SSRs and expressed sequence tag (EST), restriction fragment
length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPD) have
been developed [
110
,
111
]. Through SSR analysis, Abraha, et al. [
112
] identified and im-
proved some important traits in tef, including grain yield, days to maturity, panicle length
and plant height. Similarly, variability in tef accessions was identified using AFLP markers,
which can be used in seed multiplication and breeding programs [
113
]. Application of
Plants 2021,10, 628 7 of 19
these markers could play a great role in environmental stress tolerance in tef for improved
productivity. Targeting induced local lesions in genomes (TILLING) is another genetic
method used to identify small deletions or single base pair changes (mutation detection)
in specific target genes [
114
]. In tef, targeting induced local lesions in genomes (TILLING)
was used for targeting and improving valuable agronomic traits such as drought tolerance,
seed size and dwarfism [115].
4. Potential of Genome Editing Technologies for Tef Improvement
Genome editing is one of the most recently developed technologies that has great po-
tential to improve abiotic stress tolerance and boost productivity in tef. In a given genome,
DNA can be replaced, inserted or deleted at an endogenous loci through a robust genetic en-
gineering technique using sequence-specific nucleases (SSNs) [
116
]. SSNs such as CRISPR
and CRISPR-associated protein 9 (CRISPR-Cas9) [
117
120
], transcriptional activator-like
effector nuclease (TALEN) [
121
123
] and zinc finger nuclease (ZFN) [
124
,
125
] have been
implicated in rapid genome editing in recent years. In addition to these, plant scientists use
other techniques such as base editing, prime editing [
126
] and CRSIPR-Cpf1 [
127
]. Recently,
CRISPR-Cpf1 has successfully used the prime genome editing in wheat Lin, et al. [
128
]
and rice Lin, et al. [
128
], Li, et al. [
129
] genomes. These genome editing tools have been
used in model plants, but with advances in genome editing, these procedures are now cus-
tomized for wide variety of plant species and are usually specific to genotype [
130
] (Figure
2). However, to adopt advanced genetic engineering technologies in tef, there must be
a well-established transformation and regeneration system, which is currently underdevel-
oped or non-existent for underutilized crops including tef. Recent advances in transgenic
technologies have revealed promising tools for enhancing transformation and regeneration
of transgenic lines. For example, overexpression of the maize embryogenic regulator genes
baby boom (Bbm) and Wuschel2 (Wus2) has been shown to produce high transformation
frequencies in numerous previously non-transformable monocot species, including maize
inbred lines, sorghum (Sorghum bicolor (L.) Moench), sugarcane (Saccharum officinarum L.)
and indica rice (Oryza sativa ssp. indica) [
131
]. More recently, Debernardi et al. [
132
] re-
ported that expression of a fusion protein combining wheat growth-regulating factor 4
(GRF4) and its cofactor GRF-interacting factor 1 (GIF1) has been shown to substantially
increase the efficiency and speed of regeneration in wheat, triticale and rice and increase
the number of transformable wheat genotypes. These approaches have great potential for
genetic improvement of tef and other recalcitrant economically important crops.
Since its first application as a plant genome editing technique [
120
,
133
,
134
], CRISPR-
Cas has been widely applied in crop improvement programs [
135
,
136
]. Major crops
that have benefited from the CRISPR-Cas technique include rice [
32
35
], maize [
30
,
31
],
wheat [
30
,
36
] and other monocots [
38
]. In rice (Oryza sativa), the CRISPR-Cas system
has been used to enhance drought [
39
], cold [
40
] and salt [
41
,
42
] tolerance, and to boost
productivity [
39
]. Recently, in wheat, which is one of the plant species that is considered
recalcitrant to genetic transformation via the Agrobacterium method, two efficient and simple
CRISPR-Cas methods were developed [
43
45
]. Taken together, CRISPR-Cas technology
has been widely implemented in both monocots and dicots, and has great potential to
be implemented in tef improvement so that the performance of the crop against diverse
environmental stresses will be enhanced, with the ultimate goal of boosting productivity.
Plants 2021,10, 628 8 of 19
Plants 2021, 10, x FOR PEER REVIEW 8 of 19
Figure 2. A schematic view of genome editing by zinc finger nuclease (ZFN) and transcriptional
activator-like effector nuclease (TALEN) in tef. A desired gene is selected from tef and integrated
with ZFN and TALEN and then transferred to a cell through a vector, which will then introduce a
break into the double-stranded DNA and integrate the gene of interest into the host genome.
Transformed cells are used to regenerate to whole plants. (Note: the pictures used in this figure
were either taken in the author’s labs or drawn using ChemBioDraw software).
Candidate Tef Genes for CRISPR-Cas Technology
The CRISPR-Cas system has proven efficient because it uses a single guide RNA
through pairing of DNA targeting [137,138]. Targeting of DNA is essential for genome
editing across all organisms [139]. In order to edit any plant gene using the CRISPR-Cas
system, it is not necessary to integrate into the genome. For example, a guide RNA and
Figure 2.
A schematic view of genome editing by zinc finger nuclease (ZFN) and transcriptional
activator-like effector nuclease (TALEN) in tef. A desired gene is selected from tef and integrated with
ZFN and TALEN and then transferred to a cell through a vector, which will then introduce a break
into the double-stranded DNA and integrate the gene of interest into the host genome. Transformed
cells are used to regenerate to whole plants. (Note: the pictures used in this figure were either taken
in the author’s labs or drawn using ChemBioDraw software).
Candidate Tef Genes for CRISPR-Cas Technology
The CRISPR-Cas system has proven efficient because it uses a single guide RNA
through pairing of DNA targeting [
137
,
138
]. Targeting of DNA is essential for genome
editing across all organisms [
139
]. In order to edit any plant gene using the CRISPR-Cas
system, it is not necessary to integrate into the genome. For example, a guide RNA and Cas
Plants 2021,10, 628 9 of 19
can be expressed transiently in the protoplast to edit a plant genome, and the protoplast can
be regenerated into whole plant. Cas is a class II CRISPR system which is used in various
organisms as a gene editing tool [
138
,
140
]. The basic mechanism involved in CRISPR-
Cas editing is transformation to cells, followed by its integration with the host genome,
and expression, where it cuts the specific locus of interest on the chromosome. The genome
cleavage requires the Cas system, together with a single guided RNA (sgRNA): fusion of
trans-activating (tracr RNA) and CRISPR RNAs (crRNA), followed by the recognition of
the desired DNA sequence and protospacer-adjacent motifs (PAMs) (Figure 3) [138].
Plants 2021, 10, x FOR PEER REVIEW 9 of 19
Cas can be expressed transiently in the protoplast to edit a plant genome, and the proto-
plast can be regenerated into whole plant. Cas is a class II CRISPR system which is used
in various organisms as a gene editing tool [138,140]. The basic mechanism involved in
CRISPR-Cas editing is transformation to cells, followed by its integration with the host
genome, and expression, where it cuts the specific locus of interest on the chromosome.
The genome cleavage requires the Cas system, together with a single guided RNA
(sgRNA): fusion of trans-activating (tracr RNA) and CRISPR RNAs (crRNA), followed by
the recognition of the desired DNA sequence and protospacer-adjacent motifs (PAMs)
(Figure 3) [138].
Figure 3. Illustration of the CRISPR-Cas system for tef genome editing. The gene of interest is
transferred into a binary vector, which will be transferred into the target tissue (e.g., embryogenic
calli) via Agrobacterium transformation, where the CRISPR-Cas protein machinery binds and
breaks the double-stranded DNA of the gene of interest. CRISPR-edited lines will be regenerated
Figure 3.
Illustration of the CRISPR-Cas system for tef genome editing. The gene of interest is
transferred into a binary vector, which will be transferred into the target tissue (e.g., embryogenic
calli) via Agrobacterium transformation, where the CRISPR-Cas protein machinery binds and breaks
the double-stranded DNA of the gene of interest. CRISPR-edited lines will be regenerated from rthe
callus. (Note: the pictures used in this figure were either taken in the author’s labs or drawn using
ChemBioDraw software).
Plants 2021,10, 628 10 of 19
To utilize CRISPR-Cas technology in tef improvement, identification of target genes
that regulate agronomically important traits is crucial. In this review, we explored the draft
genome sequence of tef [
141
] to identify genes that are possible targets for improved yield
and abiotic stress tolerance. We reviewed the literature for genes which are negative regu-
lators of abiotic stress tolerance, and those that regulate plant height and yield attributes
in monocots, including rice, maize, wheat and finger millet, which is closely related to
tef. We then searched for homologs in tef (Table 1) from the Ensembl plant database using
CoGeBlast-comparative genomics databases [
142
]. The tef homologs were aligned with
those in other monocots using the Mega X clustlaw alignment tool [
143
,
144
]. After align-
ment, a phylogenetic tree was constructed using the Mega X maximum likelihood tool [
144
]
(
Figure 4
). It can be observed from Figure 4that the tef homologs showed maximum
bootstrap values with those of the other monocots.
Plants 2021, 10, x FOR PEER REVIEW 10 of 19
from rthe callus. (Note: the pictures used in this figure were either taken in the author’s labs or
drawn using ChemBioDraw software).
To utilize CRISPR-Cas technology in tef improvement, identification of target genes
that regulate agronomically important traits is crucial. In this review, we explored the
draft genome sequence of tef [141] to identify genes that are possible targets for improved
yield and abiotic stress tolerance. We reviewed the literature for genes which are negative
regulators of abiotic stress tolerance, and those that regulate plant height and yield attrib-
utes in monocots, including rice, maize, wheat and finger millet, which is closely related
to tef. We then searched for homologs in tef (Table 1) from the Ensembl plant database
using CoGeBlast-comparative genomics databases [142]. The tef homologs were aligned
with those in other monocots using the Mega X clustlaw alignment tool [143,144]. After
alignment, a phylogenetic tree was constructed using the Mega X maximum likelihood tool
[144] (Figure 4). It can be observed from Figure 4 that the tef homologs showed maximum
bootstrap values with those of the other monocots.
Figure 4. Phylogenetic tree of stress-resistant genes in tef and related monocots. The tree was con-
structed by using specific gene sequences downloaded from NCBI and Ensembl Plants. Bootstrap
values (1000 pseudoreplicates) are shown on the nodes of the branches.
Tef is tolerant to poor soil conditions including waterlogging and drought [145].
However, tef yield is reduced by lodging, terminal drought and diseases. Therefore, tef is
expected to benefit from CRISPR-Cas genome editing technology. The draft genome se-
quence of tef has been released [141]. Two complete homologous chromosomes with
syntenic gene pairs have been reported in the tef genome due to its allotetraploid genome.
The subgenomes are small (~300 Mb), with a low number of transposable elements (TE)
Figure 4.
Phylogenetic tree of stress-resistant genes in tef and related monocots. The tree was
constructed by using specific gene sequences downloaded from NCBI and Ensembl Plants. Bootstrap
values (1000 pseudoreplicates) are shown on the nodes of the branches.
Tef is tolerant to poor soil conditions including waterlogging and drought [
145
].
However, tef yield is reduced by lodging, terminal drought and diseases. Therefore, tef
is expected to benefit from CRISPR-Cas genome editing technology. The draft genome
sequence of tef has been released [
141
]. Two complete homologous chromosomes with
syntenic gene pairs have been reported in the tef genome due to its allotetraploid genome.
The subgenomes are small (~300 Mb), with a low number of transposable elements (TE)
and a high density of genes as compared to other polyploid grasses [
141
]. One of the major
obstacles for the targeted breeding of tef is the presence of genes in two genomes (AA
and BB: tef is allotetraploid, with 2n = 4x = 40 chromosomes). Gene redundancy poses
Plants 2021,10, 628 11 of 19
a difficulty in mutagenesis for developing lodging-resistant and semi-dwarf varieties [
146
].
This obstacle can be overcome by techniques such as targeted genome engineering and
marker assisted selection. In a plant genome, the majority of genes have variable expression
patterns; therefore, the two sub-genomes are more likely to affect agronomic traits with
different frequencies [
141
,
147
]. To utilize CRISPR-Cas technology in tef improvement,
the identification of target genes that regulate agronomically important traits is crucial.
Table 1.
Summary of genes involved in key agronomic traits of selected crops. Homologs of these genes in tef were
downloaded from the genomic database to identify potential candidate genes for CRISPR-Cas-mediated gene editing in tef.
Gene Plant Name Accession Number Reference
Plant Height
KO2Oryza sativa Japonica AY660664 [148]
GA regulatory factor-like (GRF) mRNA Zea mays KJ466125 [149]
growth-regulating factor 10 (GRF10) Oryza sativa Indica FJ546694 [150]
GA20-oxidase (GA20ox2) Oryza granulata EU179380 [151]
BRI1 Triticum aestivum DQ655711 [152]
Sd-1 (used in green revl) Oryza sativa KP212897.1 [70]
RHT1 Triticum aestivum FN649763 [153]
Number of Tillers and Panicle Branches
OsCKX2 Oryza sativa AB205193.1 [154]
teosinte branched1 (tb1) switchgrass AF131673.2 [155]
GSK2 Oryza sativa XM_015782085 [156]
PYL2 Oryza sativa KJ700410.1
[157]
PYL3, Oryza sativa KJ191278.1
PYL4, Oryza sativa KJ855099.1
PYL5, Oryza sativa KJ855100.1
PYL6 Oryza sativa KJ855101.1
PYL12 Oryza sativa KJ855107.1
monoculm1 MOC1 Oryza sativa Japonica KC700671.1 [158]
Grain Size
G1F1A Oryza sativa GU797949 [159]
Drought Tolerance
GhWRKY33 Gossypium hirsutum KJ825875.1 [160]
WRKY mRNA Triticum aestivum KT865879 [161]
threonine dehydratase mRNA Eleusine coracana MK573864 [162]
OsCDPK7 Oryza sativa Japonica AB042550 [163]
TaWRKY146 Triticum aestivum MF770640.1 [164]
NF-Y18 Oryza sativa Japonica HQ731479 [165]
Arginine decarboxylase (ADC) Oryza sativa Japonica CA754598.1 [166]
CIPK12 Oryza sativa Japonica EU703798 [166]
NF-YB Zea mays NM_001112582 [167]
5. Constraints and Solutions Related to CRISPR-Cas Genome Editing
The stable transfer of the transgene into the target site using CRISPR-Cas during
the transformation process may cause the insertion of plasmid DNA or unwanted genes,
which makes it a genetically modified (GM) crop. This limits the use of CRISPR-Cas
system for sustainable agriculture and biotechnology because in some countries the use of
GMOs is either tightly regulated or totally prohibited [
168
]. Although genetic segregation
is the process by which the foreign DNA can be removed, this is not applicable to some
clonally propagated plants. Moreover, in some countries, CRISPR-Cas products are still
not acceptable because foreign DNA materials are used in the process, although these
foreign materials are removed at the end [
168
]. In plants, DNA-free genome editing
has been conducted using two approaches; these are pre-assembled ribonucleoproteins
Plants 2021,10, 628 12 of 19
(RNPs) [
169
,
170
] and the delivery of a combination of guide RNA and mRNA-encoding
Cas [
43
]. However, CRISPR-Cas RNA transient expression efficiency is low, suggesting
a need for additional optimization. Following this approach, the addition of a protectant
for stabilizing RNA could prove to be a promising strategy [171].
Another major drawback of the CRISPR-Cas system is its non-specificity. In this case,
Cas cleaves DNA at non-target sites that are not complementary of single guide RNA [
172
].
This drawback impedes CRISPR-Cas potential applications, particularly when genome
alteration needs to be precise, as in the case of gene therapy. Off-target sites may not
change plant breeding as much as the chemical and physical alterations used in traditional
breeding procedures, which generate many alterations in plants [
173
]. These off-target
alterations can be removed by performing backcrossing to the original plant. However,
this takes several generations of investigation, and the improvement of the process will
be slow.
In plants, the specificity of the CRISPR-Cas system has been estimated by deliberate
non-target investigation [
174
]. For RNPs, non-target alterations were hardly recognized
by thorough sequencing, indicating that RNPs enhance the specificity of the editing sys-
tem [
172
]. However, no study has been reported on Cas specificity in plants. Several
impartial strategies which include Digenome-seq, high-throughput genome-wide translo-
cation sequencing (HTGTS), genome-wide unbiased identification of double stranded
breaks (DSBs) enabled by sequencing (GUIDE-seq) and breaks labeling, enrichment on
streptavidin, and sequencing (BLESS) have been used to investigate non-specific changes
in human cells [
175
178
], and these strategies need to be administered in plants to evaluate
Cas specificity at the genome level. The need for improving its specificity is a major chal-
lenge for CRISPR-Cas genome editing, which requires attention. Various strategies have
been established for improving specificity [
179
], including high-fidelity Cas variants and
the Cas paired nickase strategy [180182].
6. Conclusions
Climate change and global warming are expected to trigger major abiotic stresses,
which are expected to reduce crop yields and ultimately lead to food shortages in the fore-
seeable future. Since agricultural crops fulfill most of the world’s food supply, it should
be the topmost priority of plant biologists to take concrete measures to cope with climate
change and the forecasted food shortages. Climate change and global warming are mani-
fested by abiotic stress factors that could reduce crop productivity. The goal of this review
was to provide an insight on the potential of advanced tools such as CRISPR-Cas for use by
plant biologists in order to improve stress resilience, modify plant architecture and improve
productivity. Application of this cutting-edge technology in underutilized/orphan crops
such as tef will provide several benefits. It is expected to improve food security in the Horn
of Africa, a region which is very vulnerable to the negative impact of climate change,
and which has been experiencing frequent food insecurity and adding to the global refugee
crisis. It will also enhance the acceptance of tef as a healthy and nutritious grain, which
will play a role in reducing micronutrient deficiency.
Author Contributions:
M.N. and A.L.-O. conceived the review; M.N. wrote the draft of the manuscript;
A.L.-O., M.S., Z.T., G.B., A.L.K. and S.A. contributed and edited the manuscript. All authors have
read and agreed to the published version of the manuscript.
Funding:
This manuscript was support by the University of North Carolina at Greensboro (Grant #
133504 to A.L.-O.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: All the authors declare no conflict of interest.
Plants 2021,10, 628 13 of 19
References
1. Cheng, A. Shaping a sustainable food future by rediscovering long-forgotten ancient grains. Plant Sci. 2018,269, 136–142. [CrossRef]
2.
Ray, D.K.; Gerber, J.S.; MacDonald, G.K.; West, P.C. Climate variation explains a third of global crop yield variability. Nat. Commun.
2015,6, 1–9. [CrossRef]
3.
Cochrane, L.; Bekele, Y.W. Average crop yield (2001–2017) in Ethiopia: Trends at national, regional and zonal levels. Data Brief
2018,16, 1025. [CrossRef] [PubMed]
4. CSA. Agricultural Sample Survey 2015/2016; CSA: Addis Ababa, Ethiopia, 2016.
5.
Tadele, Z.; Assefa, K. Increasing food production in Africa by boosting the productivity of understudied crops. Agronomy
2012
,2,
240–283. [CrossRef]
6.
El-Alfy, T.S.; Ezzat, S.M.; Sleem, A.A. Chemical and biological study of the seeds of Eragrostis tef (Zucc.) Trotter. Nat. Prod. Res.
2012,26, 619–629. [CrossRef]
7.
Spaenij-Dekking, L.; Kooy-Winkelaar, Y.; Koning, F. The Ethiopian cereal tef in celiac disease. N. Engl. J. Med.
2005
,353, 1748–1749.
[CrossRef]
8. Saturni, L.; Ferretti, G.; Bacchetti, T. The gluten-free diet: Safety and nutritional quality. Nutrients 2010,2, 16–34. [CrossRef]
9.
Gujral, N.; Freeman, H.J.; Thomson, A.B. Celiac disease: Prevalence, diagnosis, pathogenesis and treatment. World J. Gastroen-
terol. WJG 2012,18, 6036. [CrossRef] [PubMed]
10.
Yami, A. 17. Tef Straw: A Valuable Feed Resource to Improve Animal Production and Productivity; Tef Improvement: Bern, Switzerland,
2013; Volume 233.
11.
Assefa, K.; Yu, J.K.; Zeid, M.; Belay, G.; Tefera, H.; Sorrells, M. Breeding tef [Eragrostis tef (Zucc.) Trotter]: Conventional and
molecular approaches. Plant Breed. 2011,130, 1–9. [CrossRef]
12.
Fang, X.; Li, Y.; Nie, J.; Wang, C.; Huang, K.; Zhang, Y.; Zhang, Y.; She, H.; Liu, X.; Ruan, R. Effects of nitrogen fertilizer and
planting density on the leaf photosynthetic characteristics, agronomic traits and grain yield in common buckwheat (Fagopyrum es-
culentum M.). Field Crops Res. 2018,219, 160–168. [CrossRef]
13.
Zhao, H.; Mo, Z.; Lin, Q.; Pan, S.; Duan, M.; Tian, H.; Wang, S.; Tang, X. Relationships between grain yield and agronomic traits of
rice in southern China. Chil. J. Agric. Res. 2020,80, 72–79. [CrossRef]
14.
Belete, T.; Mathewos, T.; Daba, G. Correlation of yield and yield related traits of Tef (Eragrostis tef (Zucc.) Trotter) varieties
in Ethiopia. J. Genet. Environ. Resour. Conserv. 2020,8, 35–39.
15.
Tilahun, Z.M. Effect of row spacing and nitrogen fertilizer levels on yield and yield components of rice varieties. World Sci. News
2019,116, 180–193.
16.
Arefaine, A.; Adhanom, D.; Tekeste, N. Response of Teff (Eragrostis tef (Zucc.) Trotter) to Seeding Rate and Methods of Sowing on
Yield and Yield Attributes in a Subhumid Environment, Northern Ethiopia. Int. J. Agron. 2020,2020, 1–7. [CrossRef]
17.
Haftamu, G.; Mitiku, H.; Yamoah, C. Tillage frequency, soil compaction and N-fertilizer rate effects on yield of teff (Eragrostis tef
(Zucc.) Trotter) in central zone of Tigray, Northern Ethiopia. Momona Ethiop. J. Sci. 2009,1, 82–94.
18.
Menalled, F.D. Sustainable Agriculture and Integrated Weed Management. In Weed Control: Sustainability, Hazards, and Risks
in Cropping Systems Worldwide; CRC Press: Boca Raton, FL, USA, 2018; Volume 3.
19.
Abrha, B.; Tsegay, A.; Gebrehiwot, K. Economic analysis of tef (Eragrostis tef (zucc.) trotter) yield in response to soil water, weed
and fertilizer managements in the northern highlands of Ethiopia. J. Drylands 2017,2, 675–682.
20. Rockström, J.; Falkenmark, M. Agriculture: Increase water harvesting in Africa. Nat. News 2015,519, 283. [CrossRef]
21.
Matsuura, A.; Tsuji, W.; An, P.; Inanaga, S.; Murata, K. Effect of pre-and post-heading water deficit on growth and grain yield of
four millets. Plant Prod. Sci. 2012,15, 323–331. [CrossRef]
22.
Winkel, T.; Renno, J.-F.; Payne, W. Effect of the timing of water deficit on growth, phenology and yield of pearl millet (Pennise-
tum glaucum (L.) R. Br.) grown in Sahelian conditions. J. Exp. Bot. 1997,48, 1001–1009. [CrossRef]
23.
Abraha, M.T.; Hussein, S.; Laing, M.; Assefa, K. Genetic management of drought in tef: Current status and future research
directions. Glob. J. Crop Soil Sci. Plant Breed. 2015,3, 156–161.
24.
Assefa, K.; Chanyalew, S.; Tadele, Z. Tef, Eragrostis tef (Zucc.) Trotter. Millets and Sorghum: Biology and Genetic Improvement. 2017,
pp. 226–266. Available online: https://www.researchgate.net/publication/312353091_Tef_Eragrostis_tef_Zucc_Trotter_Biology_
and_Genetic_Improvement (accessed on 19 March 2021).
25.
Blösch, R.; Plaza-Wüthrich, S.; de Reuille, P.B.; Weichert, A.; Routier-Kierzkowska, A.-L.; Cannarozzi, G.; Robinson, S.; Tadele, Z.
Panicle angle is an important factor in tef lodging tolerance. Front. Plant Sci. 2020,11, 1–12. [CrossRef]
26.
Van Delden, S.; Vos, J.; Ennos, A.; Stomph, T. Analysing lodging of the panicle bearing cereal teff (Eragrostis tef ). New Phytol.
2010
,
186, 696–707. [CrossRef] [PubMed]
27.
Ketema, S. Tef (Eragrostis Tef) Breeding, Genetic Resources, Agronomy, Utilization and Role in Ethiopian Agriculture; Institute of
Agricultural Research: Zaria, Nigeria, 1993.
28.
Jifar, H.; Tesfaye, K.; Assefa, K.; Chanyalew, S.; Tadele, Z. Semi-dwarf tef lines for high seed yield and lodging tolerance in Central
Ethiopia. Afr. Crop Sci. J. 2017,25, 419–439. [CrossRef]
29. Paff, K.; Asseng, S. A review of tef physiology for developing a tef crop model. Eur. J. Agron. 2018,94, 54–66. [CrossRef]
30.
Debbarma, J.; Sarki, Y.N.; Saikia, B.; Boruah, H.P.D.; Singha, D.L.; Chikkaputtaiah, C. Ethylene response factor (ERF) family
proteins in abiotic stresses and CRISPR–Cas9 genome editing of ERFs for multiple abiotic stress tolerance in crop plants: A review.
Mol. Biotechnol. 2019,61, 153–172. [CrossRef] [PubMed]
Plants 2021,10, 628 14 of 19
31.
Tiwari, Y.K.; Yadav, S.K. High temperature stress tolerance in maize (Zea mays L.): Physiological and molecular mechanisms.
J. Plant Biol. 2019,62, 93–102. [CrossRef]
32.
Zhou, H.; Liu, B.; Weeks, D.P.; Spalding, M.H.; Yang, B. Large chromosomal deletions and heritable small genetic changes induced
by CRISPR/Cas9 in rice. Nucleic Acids Res. 2014,42, 10903–10914. [CrossRef] [PubMed]
33.
Jiang, W.; Zhou, H.; Bi, H.; Fromm, M.; Yang, B.; Weeks, D.P. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene
modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 2013,41, e188. [CrossRef]
34.
Saika, H.; Mori, A.; Endo, M.; Toki, S. Targeted deletion of rice retrotransposon Tos17 via CRISPR/Cas9. Plant Cell Rep.
2019
,38,
455–458. [CrossRef]
35.
Lee, K.; Eggenberger, A.L.; Banakar, R.; McCaw, M.E.; Zhu, H.; Main, M.; Kang, M.; Gelvin, S.B.; Wang, K. CRISPR/Cas9-mediated
targeted T-DNA integration in rice. Plant Mol. Biol. 2019,99, 317–328. [CrossRef]
36.
Dayani, S.; Sabzalian, M.R.; Mazaheri-Tirani, M. CRISPR/Cas9 Genome Editing in Bread Wheat (Triticum aestivum L.) Genetic
Improvement. In Advances in Plant Breeding Strategies: Cereals; Springer: Berlin/Heidelberg, Germany, 2019; pp. 453–469.
37.
Jaganathan, D.; Ramasamy, K.; Sellamuthu, G.; Jayabalan, S.; Venkataraman, G. CRISPR for crop improvement: An update review.
Front. Plant Sci. 2018,9, 985. [CrossRef] [PubMed]
38.
Basso, M.F.; Ferreira, P.C.G.; Kobayashi, A.K.; Harmon, F.G.; Nepomuceno, A.L.; Molinari, H.B.C.; Grossi-de-Sa, M.F. Micro
RNA s and new biotechnological tools for its modulation and improving stress tolerance in plants. Plant Biotechnol. J.
2019
,17,
1482–1500. [CrossRef] [PubMed]
39.
Shi, J.; Gao, H.; Wang, H.; Lafitte, H.R.; Archibald, R.L.; Yang, M.; Hakimi, S.M.; Mo, H.; Habben, J.E. ARGOS 8 variants generated by
CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnol. J.
2017
,15, 207–216. [CrossRef] [PubMed]
40.
Shen, C.; Que, Z.; Xia, Y.; Tang, N.; Li, D.; He, R.; Cao, M. Knock out of the annexin gene OsAnn3 via CRISPR/Cas9-mediated
genome editing decreased cold tolerance in rice. J. Plant Biol. 2017,60, 539–547. [CrossRef]
41.
Zhang, A.; Liu, Y.; Wang, F.; Li, T.; Chen, Z.; Kong, D.; Bi, J.; Zhang, F.; Luo, X.; Wang, J. Enhanced rice salinity tolerance via
CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene. Mol. Breed. 2019,39, 47. [CrossRef] [PubMed]
42.
Sadanandom, A.; Srivastava, A.K.; Zhang, C. Targeted mutagenesis of the SUMO protease, Overly Tolerant to Salt1 in rice through
CRISPR/Cas9-mediated genome editing reveals a major role of this SUMO protease in salt tolerance. BioRxiv 2019. [CrossRef]
43.
Zhang, Y.; Liang, Z.; Zong, Y.; Wang, Y.; Liu, J.; Chen, K.; Qiu, J.-L.; Gao, C. Efficient and transgene-free genome editing in wheat
through transient expression of CRISPR/Cas9 DNA or RNA. Nat. Commun. 2016,7, 1–8. [CrossRef]
44.
Wang, W.; Akhunova, A.; Chao, S.; Akhunov, E. Optimizing multiplex CRISPR/Cas9-based genome editing for wheat. BioRxiv
2016. [CrossRef]
45.
Howells, R.M.; Craze, M.; Bowden, S.; Wallington, E.J. Efficient generation of stable, heritable gene edits in wheat using
CRISPR/Cas9. BMC Plant Biol. 2018,18, 215. [CrossRef] [PubMed]
46.
Berry, P.; Sterling, M.; Spink, J.; Baker, C.; Sylvester-Bradley, R.; Mooney, S.; Tams, A.; Ennos, A. Understanding and reducing
lodging in cereals. Adv. Agron. 2004,84, 215–269.
47.
Dagnaw, H. Influence of Nitrogen Fertilizer Rates and Varieties on Grain Yield, Grain Nutrition and Injera Sensory Quality of Tef
[Eragrostis tef (Zucc.) Trotter] Varieties. Master ’s Thesis, Adis Ababa University, Addis Ababa, Ethiopia, 2018.
48. Sterling, M.; Baker, C.; Berry, P.; Wade, A. An experimental investigation of the lodging of wheat. Agric. For. Meteorol. 2003,119,
149–165. [CrossRef]
49.
Merchuk-Ovnat, L.; Bimro, J.; Yaakov, N.; Kutsher, Y.; Amir-Segev, O.; Reuveni, M. In-Depth Field Characterization of Teff
[Eragrostis tef (Zucc.) Trotter] Variation: From Agronomic to Sensory Traits. Agronomy 2020,10, 1107. [CrossRef]
50.
Assefa, K.; Cannarozzi, G.; Girma, D.; Kamies, R.; Chanyalew, S.; Plaza-Wüthrich, S.; Blösch, R.; Rindisbacher, A.; Rafudeen, S.;
Tadele, Z. Genetic diversity in tef [Eragrostis tef (Zucc.) Trotter]. Front. Plant Sci. 2015,6, 177. [CrossRef] [PubMed]
51.
Wu, W.; Ma, B.L. A new method for assessing plant lodging and the impact of management options on lodging in canola crop
production. Sci. Rep. 2016,6, 1–17. [CrossRef] [PubMed]
52.
Mariani, L.; Ferrante, A. Agronomic management for enhancing plant tolerance to abiotic stresses—Drought, salinity, hypoxia,
and lodging. Horticulturae 2017,3, 52. [CrossRef]
53. Epstein, E. The anomaly of silicon in plant biology. Proc. Natl. Acad. Sci. USA 1994,91, 11–17. [CrossRef]
54.
Ma, J.F. Role of silicon in enhancing the resistance of plants to biotic and abiotic stresses. Soil Sci. Plant Nutr.
2004
,50, 11–18. [CrossRef]
55.
De Carvalho, D.D.; Costa, F.T.; Duran, N.; Haun, M. Cytotoxic activity of violacein in human colon cancer cells. Toxicol. In Vitro
2006,20, 1514–1521. [CrossRef]
56.
Liang, Y.; Nikolic, M.; Bélanger, R.; Gong, H.; Song, A. Silicon in Agriculture; Springer: Dordrecht, The Netherlands, 2015;
Volume 10, pp. 978–994.
57.
Ligaba-Osena, A.; Guo, W.; Choi, S.C.; Limmer, M.A.; Seyfferth, A.L.; Hankoua, B.B. Silicon enhances biomass and grain yield
in an ancient crop tef [Eragrostis tef (Zucc.) Trotter]. Front. Plant Sci. 2020,11, 608503. [CrossRef]
58. Deshmukh, R.K.; Ma, J.F.; Bélanger, R.R. Role of silicon in plants. Front. Plant Sci. 2017,8, 1858. [CrossRef]
59.
Jöst, M.; Esfeld, K.; Burian, A.; Cannarozzi, G.; Chanyalew, S.; Kuhlemeier, C.; Assefa, K.; Tadele, Z. Semi-dwarfism and lodging
tolerance in tef (Eragrostis tef ) is linked to a mutation in the α-Tubulin 1 gene. J. Exp. Bot. 2015,66, 933–944. [CrossRef]
60.
Cannarozzi, G.; Chanyalew, S.; Assefa, K.; Bekele, A.; Blösch, R.; Weichert, A.; Klauser, D.; Plaza-Wüthrich, S.; Esfeld, K.; Jöst, M.
Technology generation to dissemination: Lessons learned from the tef improvement project. Euphytica
2018
,214, 1–20. [CrossRef]
Plants 2021,10, 628 15 of 19
61.
Rademacher, W. Growth retardants: Effects on gibberellin biosynthesis and other metabolic pathways. Annu. Rev. Plant Biol.
2000,51, 501–531. [CrossRef] [PubMed]
62.
Sanvicente, P.; Lazarevitch, S.; Blouet, A.; Guckert, A. Morphological and anatomical modifications in winter barley culm after
late plant growth regulator treatment. Eur. J. Agron. 1999,11, 45–51. [CrossRef]
63. Hedden, P. The genes of the Green Revolution. Trends Genet. 2003,19, 5–9. [CrossRef]
64.
Itoh, H.; Ueguchi-Tanaka, M.; Sentoku, N.; Kitano, H.; Matsuoka, M.; Kobayashi, M. Cloning and functional analysis of two
gibberellin 3
β
-hydroxylase genes that are differently expressed during the growth of rice. Proc. Natl. Acad. Sci. USA
2001
,98,
8909–8914. [CrossRef] [PubMed]
65.
Monna, L.; Kitazawa, N.; Yoshino, R.; Suzuki, J.; Masuda, H.; Maehara, Y.; Tanji, M.; Sato, M.; Nasu, S.; Minobe, Y. Positional
cloning of rice semidwarfing gene, sd-1: Rice “green revolution gene” encodes a mutant enzyme involved in gibberellin synthesis.
DNA Res. 2002,9, 11–17. [CrossRef] [PubMed]
66.
Sasaki, A.; Ashikari, M.; Ueguchi-Tanaka, M.; Itoh, H.; Nishimura, A.; Swapan, D.; Ishiyama, K.; Saito, T.; Kobayashi, M.;
Khush, G.S. Green revolution: A mutant gibberellin-synthesis gene in rice. Nature 2002,416, 701. [CrossRef]
67.
Hong, Z.; Ueguchi-Tanaka, M.; Umemura, K.; Uozu, S.; Fujioka, S.; Takatsuto, S.; Yoshida, S.; Ashikari, M.; Kitano, H.; Matsuoka,
M. A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450.
Plant Cell 2003,15, 2900–2910. [CrossRef] [PubMed]
68.
Multani, D.S.; Briggs, S.P.; Chamberlin, M.A.; Blakeslee, J.J.; Murphy, A.S.; Johal, G.S. Loss of an MDR transporter in compact
stalks of maize br2 and sorghum dw3 mutants. Science 2003,302, 81–84. [CrossRef]
69.
Asano, K.; Hirano, K.; Ueguchi-Tanaka, M.; Angeles-Shim, R.B.; Komura, T.; Satoh, H.; Kitano, H.; Matsuoka, M.; Ashikari, M.
Isolation and characterization of dominant dwarf mutants, Slr1-d, in rice. Mol. Genet. Genom. 2009,281, 223–231. [CrossRef]
70.
Spielmeyer, W.; Ellis, M.H.; Chandler, P.M. Semidwarf (sd-1),“green revolution” rice, contains a defective gibberellin 20-oxidase
gene. Proc. Natl. Acad. Sci. USA 2002,99, 9043–9048. [CrossRef] [PubMed]
71.
Muangprom, A.; Thomas, S.G.; Sun, T.-p.; Osborn, T.C. A novel dwarfing mutation in a green revolution gene from Brassica rapa.
Plant Physiol. 2005,137, 931–938. [CrossRef]
72.
Peng, J.; Richards, D.E.; Hartley, N.M.; Murphy, G.P.; Devos, K.M.; Flintham, J.E.; Beales, J.; Fish, L.J.; Worland, A.J.; Pelica, F.
‘Green revolution’genes encode mutant gibberellin response modulators. Nature 1999,400, 256. [CrossRef] [PubMed]
73.
Wang, Y.; Chen, L.; Du, Y.; Yang, Z.; Condon, A.G.; Hu, Y.-G. Genetic effect of dwarfing gene Rht13 compared with Rht-D1b on
plant height and some agronomic traits in common wheat (Triticum aestivum L.). Field Crops Res. 2014,162, 39–47. [CrossRef]
74.
Itoh, H.; Tatsumi, T.; Sakamoto, T.; Otomo, K.; Toyomasu, T.; Kitano, H.; Ashikari, M.; Ichihara, S.; Matsuoka, M. A rice semi-dwarf gene,
Tan-Ginbozu (D35), encodes the gibberellin biosynthesis enzyme, ent-kaurene oxidase. Plant Mol. Biol. 2004,54, 533–547. [CrossRef]
75.
Hilley, J.L.; Weers, B.D.; Truong, S.K.; McCormick, R.F.; Mattison, A.J.; McKinley, B.A.; Morishige, D.T.; Mullet, J.E. Sorghum Dw2
encodes a protein kinase regulator of stem internode length. Sci. Rep. 2017,7, 1–13. [CrossRef] [PubMed]
76.
Yamaguchi, M.; Fujimoto, H.; Hirano, K.; Araki-Nakamura, S.; Ohmae-Shinohara, K.; Fujii, A.; Tsunashima, M.; Song, X.J.; Ito, Y.;
Nagae, R. Sorghum Dw1, an agronomically important gene for lodging resistance, encodes a novel protein involved in cell
proliferation. Sci. Rep. 2016,6, 1–11. [CrossRef] [PubMed]
77.
Mickelbart, M.V.; Hasegawa, P.M.; Bailey-Serres, J. Genetic mechanisms of abiotic stress tolerance that translate to crop yield
stability. Nat. Rev. Genet. 2015,16, 237–251. [CrossRef] [PubMed]
78.
Duque, A.S.; de Almeida, A.M.; da Silva, A.B.; da Silva, J.M.; Farinha, A.P.; Santos, D.; Fevereiro, P.; de Sousa Araújo, S. Abiotic
stress responses in plants: Unraveling the complexity of genes and networks to survive. In Abiotic Stress-Plant Responses and
Applications in Agriculture; IntechOpen: London, UK, 2013; pp. 49–101. [CrossRef]
79.
Ayele, M.; Blum, A.; Nguyen, H.T. Diversity for osmotic adjustment and root depth in tef [Eragrostis tef (Zucc.) Trotter]. Euphytica
2001,121, 237–249. [CrossRef]
80.
Araya, A.; Stroosnijder, L.; Girmay, G.; Keesstra, S. Crop coefficient, yield response to water stress and water productivity of teff
(Eragrostis tef (Zucc.). Agric. Water Manag. 2011,98, 775–783. [CrossRef]
81.
Plaza-Wüthrich, S.; Blösch, R.; Rindisbacher, A.; Cannarozzi, G.; Tadele, Z. Gibberellin deficiency confers both lodging and
drought tolerance in small cereals. Front. Plant Sci. 2016,7, 643. [CrossRef]
82.
Kusaka, M.; Lalusin, A.G.; Fujimura, T. The maintenance of growth and turgor in pearl millet (Pennisetum glaucum [L.] Leeke)
cultivars with different root structures and osmo-regulation under drought stress. Plant Sci. 2005,168, 1–14. [CrossRef]
83.
Farooq, M.; Wahid, A.; Kobayashi, N.; Fujita, D.; Basra, S. Plant drought stress: Effects, mechanisms and management. In Sustain-
able Agriculture; Springer: Berlin/Heidelberg, Germany, 2009; pp. 153–188.
84.
Merrill, S.D.; Tanaka, D.L.; Hanson, J.D. Root length growth of eight crop species in Haplustoll soils. Soil Sci. Soc. Am. J.
2002
,66,
913–923. [CrossRef]
85.
Renton, M.; Chauhan, B.S. Modelling crop-weed competition: Why, what, how and what lies ahead? Crop Prot.
2017
,95, 101–108. [CrossRef]
86.
Andrew, I.; Storkey, J.; Sparkes, D. A review of the potential for competitive cereal cultivars as a tool in integrated weed
management. Weed Res. 2015,55, 239–248. [CrossRef]
87.
Hansen, P.K.; Kristensen, K.; Willas, J. A weed suppressive index for spring barley (Hordeum vulgare) varieties. Weed Res.
2008
,48,
225–236. [CrossRef]
88.
Gebrehiwot, H.G.; Aune, J.B.; Netland, J.; Eklo, O.M.; Torp, T.; Brandsæter, L.O. Weed-Competitive Ability of Teff (Eragrostis tef
(Zucc.) Trotter) Varieties. Agronomy 2020,10, 108. [CrossRef]
Plants 2021,10, 628 16 of 19
89.
Laizer, H.C.; Chacha, M.N.; Ndakidemi, P.A. Farmers’ knowledge, perceptions and practices in managing weeds and insect pests
of common bean in Northern Tanzania. Sustainability 2019,11, 4076. [CrossRef]
90.
Dezfulian, M.H.; Foreman, C.; Jalili, E.; Pal, M.; Dhaliwal, R.K.; Roberto, D.K.A.; Imre, K.M.; Kohalmi, S.E.; Crosby, W.L.
Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and
Arabidopsis development. BMC Plant Biol. 2017,17, 71. [CrossRef]
91.
Singh, S.; Kumar, V.; Dhanjal, D.S.; Singh, J. Herbicides and Plant Growth Regulators: Current Developments and Future Challenges.
In Natural Bioactive Products in Sustainable Agriculture; Singh, J., Yadav, A.N., Eds.; Springer: Singapore, 2020. [CrossRef]
92.
Küpper, A.; Borgato, E.A.; Patterson, E.L.; Netto, A.G.; Nicolai, M.; Carvalho, S.J.P.d.; Nissen, S.J.; Gaines, T.A.; Christoffoleti, P.J.
Multiple Resistance to Glyphosate and Acetolactate Synthase Inhibitors in Palmer Amaranth (Amaranthus palmeri) Identified
in Brazil. Weed Sci. 2017,65, 317–326. [CrossRef]
93.
Xue, W.; Xing, Y.; Weng, X.; Zhao, Y.; Tang, W.; Wang, L.; Zhou, H.; Yu, S.; Xu, C.; Li, X. Natural variation in Ghd7 is an important
regulator of heading date and yield potential in rice. Nat. Genet. 2008,40, 761–767. [CrossRef]
94.
Guillen-Portal, F.R.; Stougaard, R.N.; Xue, Q.; Eskridge, K.M. Compensatory mechanisms associated with the effect of spring
wheat seed size on wild oat competition. Crop Sci. 2006,46, 935–945. [CrossRef]
95.
Gupta, P.K.; Rustgi, S.; Kumar, N. Genetic and molecular basis of grain size and grain number and its relevance to grain
productivity in higher plants. Genome 2006,49, 565–571. [CrossRef]
96.
Huang, M.; Zou, Y.-B.; Jiang, P.; Bing, X.; Md, I.; Ao, H.-J. Relationship between grain yield and yield components in super hybrid
rice. Agric. Sci. China 2011,10, 1537–1544. [CrossRef]
97.
Yao, F.; Huang, J.; Cui, K.; Nie, L.; Xiang, J.; Liu, X.; Wu, W.; Chen, M.; Peng, S. Agronomic performance of high-yielding rice
variety grown under alternate wetting and drying irrigation. Field Crops Res. 2012,126, 16–22. [CrossRef]
98.
Terao, T.; Nagata, K.; Morino, K.; Hirose, T. A gene controlling the number of primary rachis branches also controls the vascular
bundle formation and hence is responsible to increase the harvest index and grain yield in rice. Theor. Appl. Genet.
2010
,120,
875–893. [CrossRef]
99.
Zhang, Y.-C.; Yu, Y.; Wang, C.-Y.; Li, Z.-Y.; Liu, Q.; Xu, J.; Liao, J.-Y.; Wang, X.-J.; Qu, L.-H.; Chen, F. Overexpression of microRNA
OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nat. Biotechnol.
2013
,31, 848–852.
[CrossRef] [PubMed]
100.
Huang, X.; Qian, Q.; Liu, Z.; Sun, H.; He, S.; Luo, D.; Xia, G.; Chu, C.; Li, J.; Fu, X. Natural variation at the DEP1 locus enhances
grain yield in rice. Nat. Genet. 2009,41, 494–497. [CrossRef]
101.
Miura, K.; Ikeda, M.; Matsubara, A.; Song, X.-J.; Ito, M.; Asano, K.; Matsuoka, M.; Kitano, H.; Ashikari, M. OsSPL14 promotes
panicle branching and higher grain productivity in rice. Nat. Genet. 2010,42, 545–549. [CrossRef]
102. Assefa, K.; Chanyalew, S.; Metaferia, G. Conventional and Molecular Tef Breeding; Tef Improvement: Bern, Switzerland, 2011; Volume 33.
103.
Tadele, Z.; Ferede Haile, B.; Abreha, E.; Assefa, K.; Chanyalew, S.; Mekbib, F. Morpho-Physiologic, Genotype X Environment Interaction and
In Vitro Evaluation for Drought Tolerance in Tef Eragrostis tef (Zucc.) Trotter, Ethiopia; Haramaya University: Haromaya, Ethiopia, 2018.
104. Berehe, T. Breakthrough in tef breeding technique. FAO Inf. Bull. Cerealimprovement Prod. Near East Proj. 1975,3, 11–23.
105.
Belay, G.; Tefera, H.; Getachew, A.; Assefa, K.; Metaferia, G. Highly client-oriented breeding with farmer participation
in the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]. Afr. J. Agric. Res. 2008,3, 22–28.
106.
Belay, G.; Tefera, H.; Tadesse, B.; Metaferia, G.; Jarra, D.; Tadesse, T. Participatory variety selection in the Ethiopian cereal tef
(Eragrostis tef ). Exp. Agric. 2006,42, 91–101. [CrossRef]
107.
Chanyalew, S.; Assefa, K.; Tadele, Z. Tef [Eragrostis tef (Zucc.) Trotter] Breeding. In Advances in Plant Breeding Strategies: Cereals;
Springer: Berlin/Heidelberg, Germany, 2019; pp. 373–403.
108.
Ibitoye, D.; Akin-Idowu, P. Marker-assisted-selection (MAS): A fast track to increase genetic gain in horticultural crop breeding.
Afr. J. Biotechnol. 2010,9, 8889–8895.
109. Jiang, G.-L. Molecular markers and marker-assisted breeding in plants. Plant Breed. Lab. Fields 2013, 45–83. [CrossRef]
110.
Yu, J.-K.; Sun, Q.; Rota, M.L.; Edwards, H.; Tefera, H.; Sorrells, M.E. Expressed sequence tag analysis in tef (Eragrostis tef (Zucc.)
Trotter). Genome 2006,49, 365–372. [CrossRef]
111.
Bai, G.; Ayele, M.; Tefera, H.; Nguyen, H.T. Amplified fragment length polymorphism analysis of tef [Eragrostis tef (Zucc.) Trotter].
Crop Sci. 1999,39, 819–824. [CrossRef]
112.
Abraha, M.T.; Shimelis, H.; Laing, M.; Assefa, K.; Amelework, B. Assessment of the genetic relationship of tef (Eragrostis tef )
genotypes using SSR markers. S. Afr. J. Bot. 2016,105, 106–110. [CrossRef]
113.
Ayele, M.; Nguyen, H. Evaluation of amplified fragment length polymorphism markers in tef, Eragrostis tef (Zucc.) Trotter,
and related species. Plant Breed. 2000,119, 403–409. [CrossRef]
114.
Kashtwari, M.; Wani, A.A.; Rather, R.N. TILLING: An alternative path for crop improvement. J. Crop Improv.
2019
,33, 83–109. [CrossRef]
115. Tadele, Z. Orphan crops: Their importance and the urgency of improvement. Planta 2019,250, 677–694. [CrossRef]
116. Voytas, D.F. Plant Genome Engineering with Sequence-Specific Nucleases. Annu. Rev. Plant Biol. 2013,64, 327–350. [CrossRef]
117.
Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J.A.; Charpentier, E. A programmable dual-RNA–guided DNA
endonuclease in adaptive bacterial immunity. Science 2012,337, 816–821. [CrossRef]
118.
Cong, L.; Ran, F.A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P.D.; Wu, X.; Jiang, W.; Marraffini, L.A. Multiplex genome
engineering using CRISPR/Cas systems. Science 2013,339, 819–823. [CrossRef]
Plants 2021,10, 628 17 of 19
119.
Feng, Z.; Zhang, B.; Ding, W.; Liu, X.; Yang, D.-L.; Wei, P.; Cao, F.; Zhu, S.; Zhang, F.; Mao, Y. Efficient genome editing in plants
using a CRISPR/Cas system. Cell Res. 2013,23, 1229. [CrossRef]
120.
Shan, Q.; Wang, Y.; Li, J.; Zhang, Y.; Chen, K.; Liang, Z.; Zhang, K.; Liu, J.; Xi, J.J.; Qiu, J.-L. Targeted genome modification of crop
plants using a CRISPR-Cas system. Nat. Biotechnol. 2013,31, 686. [CrossRef] [PubMed]
121.
Christian, M.; Cermak, T.; Doyle, E.L.; Schmidt, C.; Zhang, F.; Hummel, A.; Bogdanove, A.J.; Voytas, D.F. Targeting DNA
double-strand breaks with TAL effector nucleases. Genetics 2010,186, 757–761. [CrossRef] [PubMed]
122.
Zhang, Y.; Zhang, F.; Li, X.; Baller, J.A.; Qi, Y.; Starker, C.G.; Bogdanove, A.J.; Voytas, D.F. Transcription activator-like effector
nucleases enable efficient plant genome engineering. Plant Physiol. 2013,161, 20–27. [CrossRef] [PubMed]
123.
Shan, Q.; Wang, Y.; Chen, K.; Liang, Z.; Li, J.; Zhang, Y.; Zhang, K.; Liu, J.; Voytas, D.F.; Zheng, X. Rapid and efficient gene
modification in rice and Brachypodium using TALENs. Mol. Plant 2013,6, 1365–1368. [CrossRef] [PubMed]
124.
Zhang, F.; Maeder, M.L.; Unger-Wallace, E.; Hoshaw, J.P.; Reyon, D.; Christian, M.; Li, X.; Pierick, C.J.; Dobbs, D.; Peterson, T.
High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc. Natl. Acad. Sci. USA
2010
,107,
12028–12033. [CrossRef]
125.
Sander, J.D.; Dahlborg, E.J.; Goodwin, M.J.; Cade, L.; Zhang, F.; Cifuentes, D.; Curtin, S.J.; Blackburn, J.S.; Thibodeau-Beganny, S.; Qi, Y.
Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat. Methods
2011
,8, 67. [CrossRef] [PubMed]
126.
Jiang, Z.; Hong, X.; Zhang, S.; Yao, R.; Xiao, Y. CRISPR base editing and prime editing: DSB and template-free editing systems for
bacteria and plants. Synth. Syst. Biotechnol. 2020,5, 277–292.
127.
Alok, A.; Sandhya, D.; Jogam, P.; Rodrigues, V.; Bhati, K.K.; Sharma, H.; Kumar, J. The rise of the CRISPR/Cpf1 system for
efficient genome editing in plants. Front. Plant Sci. 2020,11, 264. [CrossRef] [PubMed]
128.
Lin, Q.; Zong, Y.; Xue, C.; Wang, S.; Jin, S.; Zhu, Z.; Wang, Y.; Anzalone, A.V.; Raguram, A.; Doman, J.L. Prime genome editing
in rice and wheat. Nat. Biotechnol. 2020,38, 582–585. [CrossRef]
129.
Li, S.; Zhang, X.; Wang, W.; Guo, X.; Wu, Z.; Du, W.; Zhao, Y.; Xia, L. Expanding the scope of CRISPR/Cpf1-mediated genome
editing in rice. Mol. Plant 2018,11, 995–998. [CrossRef]
130.
Mohanta, T.K.; Bashir, T.; Hashem, A.; Abd_Allah, E.F.; Bae, H. Genome editing tools in plants. Genes
2017
,8, 399. [CrossRef] [PubMed]
131.
Lowe, K.; Wu, E.; Wang, N.; Hoerster, G.; Hastings, C.; Cho, M.-J.; Scelonge, C.; Lenderts, B.; Chamberlin, M.; Cushatt, J. Morphogenic
regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 2016,28, 1998–2015. [CrossRef] [PubMed]
132.
Debernardi, J.M.; Tricoli, D.M.; Ercoli, M.F.; Hayta, S.; Ronald, P.; Palatnik, J.F.; Dubcovsky, J. A GRF–GIF chimeric protein
improves the regeneration efficiency of transgenic plants. Nat. Biotechnol. 2020,38, 1274–1279. [CrossRef]
133.
Li, J.-F.; Norville, J.E.; Aach, J.; McCormack, M.; Zhang, D.; Bush, J.; Church, G.M.; Sheen, J. Multiplex and homologous
recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol.
2013,31, 688. [CrossRef]
134.
Nekrasov, V.; Staskawicz, B.; Weigel, D.; Jones, J.D.; Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana
using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 2013,31, 691. [CrossRef] [PubMed]
135.
Paul, J.W.; Qi, Y. CRISPR/Cas9 for plant genome editing: Accomplishments, problems and prospects. Plant Cell Rep.
2016
,35,
1417–1427. [CrossRef]
136.
Demirci, Y.; Zhang, B.; Unver, T. CRISPR/Cas9: An RNA-guided highly precise synthetic tool for plant genome editing.
J. Cell. Physiol. 2018,233, 1844–1859. [CrossRef] [PubMed]
137.
Liang, G.; Zhang, H.; Lou, D.; Yu, D. Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing. Sci. Rep.
2016,6, 1–8. [CrossRef]
138.
Ran, F.A.; Hsu, P.D.; Wright, J.; Agarwala, V.; Scott, D.A.; Zhang, F. Genome engineering using the CRISPR-Cas9 system.
Nat. Protoc. 2013,8, 2281. [CrossRef]
139.
Samanta, M.K.; Dey, A.; Gayen, S. CRISPR/Cas9: An advanced tool for editing plant genomes. Transgenic Res.
2016
,25, 561–573.
[CrossRef]
140.
Fu, Y.; Sander, J.D.; Reyon, D.; Cascio, V.M.; Joung, J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.
Nat. Biotechnol. 2014,32, 279. [CrossRef] [PubMed]
141.
VanBuren, R.; Wai, C.M.; Wang, X.; Pardo, J.; Yocca, A.E.; Wang, H.; Chaluvadi, S.R.; Han, G.; Bryant, D.; Edger, P.P. Exceptional subgenome
stability and functional divergence in the allotetraploid Ethiopian cereal teff. Nat. Commun. 2020,11, 1–11. [CrossRef] [PubMed]
142.
Joyce, B.; Baltzell, A.; Bomhoff, M.; Lyons, E. Comparative Genomics Using CoGe, Hook, Line, and Sinker. In Bioinformatics
in Aquaculture: Principles and Methods; John Wiley & Sons: Hoboken, NJ, USA, 2017. [CrossRef]
143.
Kumar, S.; Tamura, K.; Nei, M. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment.
Brief. Bioinform. 2004,5, 150–163. [CrossRef]
144.
Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing
platforms. Mol. Biol. Evol. 2018,35, 1547–1549. [CrossRef]
145. Ketema, S. Tef-Eragrostis tef (Zucc.); Bioversity International: Rome, Italy, 1997; Volume 12.
146.
Zhu, Q.; Smith, S.M.; Ayele, M.; Yang, L.; Jogi, A.; Chaluvadi, S.R.; Bennetzen, J.L. High-throughput discovery of mutations in tef
semi-dwarfing genes by next-generation sequencing analysis. Genetics 2012,192, 819–829. [CrossRef]
147.
Miller, D. Teff Grass: A new alternative. In Proceedings of the 2009 California Alfalfa & Forage Symposium and Western Seed
Conference, Reno, NV, USA, 2–4 December 2009; UC Cooperative Extension, Plant Sciences Department, University of California:
Davis, CA, USA, 2009.
Plants 2021,10, 628 18 of 19
148.
Shi, L.; Wei, X.; Adedze, Y.; Sheng, Z.; Tang, S.; Hu, P.; Wang, J. Characterization and gene cloning of the rice (Oryza sativa L.)
dwarf and narrow-leaf mutant dnl3. Genet. Mol. Res. 2016,15, 1–12. [CrossRef]
149.
Zhao, Z.; Xue, Y.; Yang, H.; Li, H.; Sun, G.; Zhao, X.; Ding, D.; Tang, J. Genome-Wide Identification of miRNAs and Their Targets
Involved in the Developing Internodes under Maize Ears by Responding to Hormone Signaling. PLoS ONE
2016
,11, e0164026.
[CrossRef] [PubMed]
150.
Zhou, M.; Gu, L.; Li, P.; Song, X.; Wei, L.; CHen, Z.; Cao, X. Degradome sequencing reveals endogenous small RNA targets in rice
(Oryza sativa L. ssp. indica). Front. Biol. 2010,5, 67–90. [CrossRef]
151.
Yang, Y.-H.; Zhang, F.-M.; Ge, S. Evolutionary rate patterns of the Gibberellin pathway genes. BMC Evol. Biol.
2009
,9, 206.
[CrossRef] [PubMed]
152.
Singla, B.; Khurana, J.P.; Khurana, P. Characterization of three somatic embryogenesis receptor kinase genes from wheat,
Triticum aestivum.Plant Cell Rep. 2008,27, 833–843. [CrossRef]
153.
Pinthus, M.; Levy, A. The relationship between the Rht 1 and Rht 2 dwarfing genes and grain weight in Triticum aestivum L. spring
wheat. Theor. Appl. Genet. 1983,66, 153–157. [CrossRef]
154.
Gouda, G.; Gupta, M.K.; Donde, R.; Mohapatra, T.; Vadde, R.; Behera, L. Marker-assisted selection for grain number and
yield-related traits of rice (Oryza sativa L.). Physiol. Mol. Biol. Plants Int. J. Funct. Plant Biol. 2020,26, 885. [CrossRef] [PubMed]
155.
Liu, Y.; Wang, W.; Yang, B.; Currey, C.; Fei, S.-Z. Functional analysis of the teosinte branched 1 gene in the tetraploid switchgrass
(Panicum virgatum L.) by CRISPR/Cas9-directed mutagenesis. BioRxiv 2020, 1–33. [CrossRef] [PubMed]
156.
Xiao, Y.; Zhang, G.; Liu, D.; Niu, M.; Tong, H.; Chu, C. GSK2 stabilizes OFP3 to suppress brassinosteroid responses in rice. Plant J.
2020,102, 1187–1201. [CrossRef]
157.
Miao, C.; Xiao, L.; Hua, K.; Zou, C.; Zhao, Y.; Bressan, R.A.; Zhu, J.-K. Mutations in a subfamily of abscisic acid receptor genes
promote rice growth and productivity. Proc. Natl. Acad. Sci. USA 2018,115, 6058–6063. [CrossRef]
158.
Xu, C.; Wang, Y.; Yu, Y.; Duan, J.; Liao, Z.; Xiong, G.; Meng, X.; Liu, G.; Qian, Q.; Li, J. Degradation of MONOCULM 1 by APC/C
TAD1 regulates rice tillering. Nat. Commun. 2012,3, 1–9. [CrossRef] [PubMed]
159.
Bhatia, D.; Joshi, S.; Das, A.; Vikal, Y.; Sahi, G.K.; Neelam, K.; Kaur, K.; Singh, K. Introgression of yield component traits in rice
(Oryza sativa ssp. indica) through interspecific hybridization. Crop Sci. 2017,57, 1557–1573. [CrossRef]
160.
Wang, N.-N.; Xu, S.-W.; Sun, Y.-L.; Liu, D.; Zhou, L.; Li, Y.; Li, X.-B. The cotton WRKY transcription factor (GhWRKY33) reduces
transgenic Arabidopsis resistance to drought stress. Sci. Rep. 2019,9, 1–13. [CrossRef] [PubMed]
161.
Satapathy, L.; Kumar, D.; Kumar, M.; Mukhopadhyay, K. Functional and DNA–protein binding studies of WRKY transcription factors
and their expression analysis in response to biotic and abiotic stress in wheat (Triticum aestivum L.). 3 Biotech 2018,8, 40. [CrossRef]
162.
Hittalmani, S.; Mahesh, H.; Shirke, M.D.; Biradar, H.; Uday, G.; Aruna, Y.; Lohithaswa, H.; Mohanrao, A. Genome and
transcriptome sequence of finger millet (Eleusine coracana (L.) Gaertn.) provides insights into drought tolerance and nutraceutical
properties. BMC Genom. 2017,18, 465. [CrossRef]
163.
Wang, Y.; Tong, X.; Qiu, J.; Li, Z.; Zhao, J.; Hou, Y.; Tang, L.; Zhang, J. A phosphoproteomic landscape of rice (Oryza sativa) tissues.
Physiol. Plant. 2017,160, 458–475. [CrossRef] [PubMed]
164.
Ma, J.; Gao, X.; Liu, Q.; Shao, Y.; Zhang, D.; Jiang, L.; Li, C. Overexpression of TaWRKY146 increases drought tolerance through
inducing stomatal closure in Arabidopsis thaliana. Front. Plant Sci. 2017,8, 2036. [CrossRef] [PubMed]
165.
Soltani Najafabadi, M. Improving rice (Oryza sativa L.) drought tolerance by suppressing a NF-YA transcription factor.
Iran. J. Biotechnol. 2012,10, 40–48.
166.
Capell, T.; Escobar, C.; Liu, H.; Burtin, D.; Lepri, O.; Christou, P. Over-expression of the oat arginine decarboxylase cDNA in trans-
genic rice (Oryza sativa L.) affects normal development patterns
in vitro
and results in putrescine accumulation in transgenic
plants. Theor. Appl. Genet. 1998,97, 246–254. [CrossRef]
167.
Li, X.-Y.; Mantovani, R.; Hooft van Huijsduijnen, R.; Andre, I.; Benoist, C.; Mathis, D. Evolutionary variation of the CCAAT-
binding transcription factor NF-Y. Nucleic Acids Res. 1992,20, 1087–1091. [CrossRef] [PubMed]
168.
Sprink, T.; Eriksson, D.; Schiemann, J.; Hartung, F. Regulatory hurdles for genome editing: Process-vs. product-based approaches
in different regulatory contexts. Plant Cell Rep. 2016,35, 1493–1506. [CrossRef]
169.
Liang, Z.; Chen, K.; Li, T.; Zhang, Y.; Wang, Y.; Zhao, Q.; Liu, J.; Zhang, H.; Liu, C.; Ran, Y. Efficient DNA-free genome editing of
bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat. Commun. 2017,8, 14261. [CrossRef] [PubMed]
170.
Woo, J.W.; Kim, J.; Kwon, S.I.; Corvalán, C.; Cho, S.W.; Kim, H.; Kim, S.-G.; Kim, S.-T.; Choe, S.; Kim, J.-S. DNA-free genome
editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat. Biotechnol. 2015,33, 1162. [CrossRef] [PubMed]
171. Latorre, A.; Latorre, A.; Somoza, Á. Modified RNAs in CRISPR/Cas9: An old trick works again. Angew. Chem. Int. Ed. 2016,55,
3548–3550. [CrossRef] [PubMed]
172.
Gerashchenkov, G.; Rozhnova, N.; Kuluev, B.; Kiryanova, O.Y.; Gumerova, G.; Knyazev, A.; Vershinina, Z.; Mikhailova, E.;
Chemeris, D.; Matniyazov, R. Design of Guide RNA for CRISPR/Cas Plant Genome Editing. Mol. Biol.
2020
,54, 24–42. [CrossRef]
173.
Salvi, S.; Druka, A.; Milner, S.G.; Gruszka, D. Induced genetic variation, TILLING and NGS-based cloning. In Biotechnological
Approaches to Barley Improvement; Springer: Berlin/Heidelberg, Germany, 2014; pp. 287–310.
174.
Zhang, D.; Li, Z.; Li, J.-F. Targeted gene manipulation in plants using the CRISPR/Cas technology. J. Genet. Genom.
2016
,43,
251–262. [CrossRef]
175.
Crosetto, N.; Mitra, A.; Silva, M.J.; Bienko, M.; Dojer, N.; Wang, Q.; Karaca, E.; Chiarle, R.; Skrzypczak, M.; Ginalski, K.
Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods
2013
,10, 361. [CrossRef]
Plants 2021,10, 628 19 of 19
176.
Frock, R.L.; Hu, J.; Meyers, R.M.; Ho, Y.-J.; Kii, E.; Alt, F.W. Genome-wide detection of DNA double-stranded breaks induced by
engineered nucleases. Nat. Biotechnol. 2015,33, 179. [CrossRef]
177.
Kim, D.; Bae, S.; Park, J.; Kim, E.; Kim, S.; Yu, H.R.; Hwang, J.; Kim, J.-I.; Kim, J.-S. Digenome-seq: Genome-wide profiling of
CRISPR-Cas9 off-target effects in human cells. Nat. Methods 2015,12, 237. [CrossRef]
178.
Tsai, S.Q.; Zheng, Z.; Nguyen, N.T.; Liebers, M.; Topkar, V.V.; Thapar, V.; Wyvekens, N.; Khayter, C.; Iafrate, A.J.; Le, L.P. GUIDE-seq
enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 2015,33, 187. [CrossRef]
179.
Hsu, P.D.; Lander, E.S.; Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell
2014
,157,
1262–1278. [CrossRef] [PubMed]
180.
Fauser, F.; Schiml, S.; Puchta, H. Both CRISPR/C as-based nucleases and nickases can be used efficiently for genome engineering
in A rabidopsis thaliana. Plant J. 2014,79, 348–359. [CrossRef] [PubMed]
181.
Slaymaker, I.M.; Gao, L.; Zetsche, B.; Scott, D.A.; Yan, W.X.; Zhang, F. Rationally engineered Cas9 nucleases with improved
specificity. Science 2016,351, 84–88. [CrossRef] [PubMed]
182.
Kleinstiver, B.P.; Pattanayak, V.; Prew, M.S.; Tsai, S.Q.; Nguyen, N.T.; Zheng, Z.; Joung, J.K. High-fidelity CRISPR–Cas9 nucleases
with no detectable genome-wide off-target effects. Nature 2016,529, 490. [CrossRef]
... However, there is an increasing interest in producing teff for human consumption outside of Ethiopia because of its outstanding nutritional value and its resilience to marginal growing conditions (AgriFutures, 2017;Numan et al., 2021). Teff grain has no gluten, low glycemic index, balanced essential amino acid content, and high mineral content when compared with other grains, and does not need to be fortified (do Nascimento, Paes, de Oliveira, Reis, & Augusta, 2018). ...
... The main factors are susceptibility to lodging, small grain size, weed competition, drought, and soil acidity (Dargo, Mekbib, & Assefa, 2016;Numan et al., 2021). The TGW of teff is generally in the range of 0.2-0.4 ...
Preprint
Full-text available
Eragrostis tef is a small annual cereal crop that naturally produces many tillers. In the past, there has been a focus on selecting for high tillering. However, having many tillers can negatively impact grain size, increase lodging, and result in uneven grain maturation. This study aimed to investigate the effectiveness of using plant growth regulators (PGRs), synthetic strigolactone (GR24) and 1-Naphthaleneacetic acid (NAA) to reduce tillering and observe their effects on grain size and yield components. The reduction in total tiller number through the application of GR24 and NAA resulted in larger grains, increased thousand-grain weight (TGW), and higher yield per panicle. The GR24 and NAA treatments led to a 12% and 8% increase in grain size and a 20% and 14% increase in TGW, respectively. Although grain yield increased with reduced tiller number, a significant reduction in tiller number could lead to lower grain yield. The application of GR24 and NAA significantly reduced the rate of tiller emergence but did not completely stop tillering. The effect of PGRs was only temporary and limited to the application period; after the treatment ceased, many tiller buds were activated and elongated. Early application of PGRs when the plant had 2-4 tillers and more frequent and longer duration of application resulted in a significant reduction in total tiller number. This suggests that large-scale application of SL and auxin on farmers' fields may not be economical. Therefore, identification of tiller inhibition genes or genetic modification leading to increased SL production or activation of SL downstream could be effective in teff.
... Ancient varieties, due to their adaptation that took place over a long time, proved to be more versatile than the main cereals and demonstrated high tolerance to various biotic and abiotic stresses [6,9]. In fact, because they are more resistant to temperature variations and require less water and fertilizer, they would reduce the impact of agriculture on the environment [10][11][12]. The removal of specific genes in crop species to improve their genetic pattern is the main cause of the enhanced resistance of some ancient grain species against biotic stresses [13]. ...
... There is evidence that some traditional food crops, such as Tef [(Eragrostis tef (Zucc. Trotter)], are particularly nutritious and are also more resilient to marginal soil and climate conditions than the major cereals [10]. Therefore, due to their unique nutritional value and phytochemical profiles, as well as their sensory characteristics, there is good potential for ancient cereals and their associated products to become part of a healthy diet [17]. ...
Article
Full-text available
According to their nutritional value, their ability to adapt to the various environmental conditions, and their versatility, cereals are among the most cultivated plants in the world. However, the ongoing climate changes subject crops to important environmental stress that for some varieties leads to high production losses. Therefore, the selection of species and varieties that are more versatile and adaptable to different environmental conditions can be important. However, the characteristics of some cereals are not completely known; this is a priority before aiming to improve their cultivation. The aim of this study is to characterize select species that are potentially suitable for local environmental conditions and that possess nutritional value. The elemental composition was assessed in different cereal species grown following intensive and organic agriculture practices. Six species were grown for this study with techniques of intensive agriculture: Triticum monococcum L., Triticum dicoccum L., Triticum aestivum L., variety Verna, Triticum durum Desf., variety Senatore Cappelli, Triticum durum Desf., variety Claudio, and Avena strigosa Schreb.; four of these were also grown following organic procedures: Triticum monococcum L., Triticum dicoccum L., Triticum aestivum L., variety Verna, and Triticum durum Desf., variety Senatore Cappelli. The study considered twenty elements, including major nutrients (Ca, K, Mg, P, and S), seven micronutrients (B, Cu, Fe, Mn, Mo, Se, and Zn), and trace elements with toxic properties (Al, Ba, Cd, Cr, Na, Rb, Sc, and Sr) that can be accumulated at the seed level. The results highlight the differences in the element concentrations in the cereal seeds in relation to the genus and species; the highest concentrations of the major nutrients appeared in T. monococcum; the concentrations were 6.9, 2.09, 7.2, and 2.9 mg/g for K, Mg, P, and S, respectively. The highest concentrations of certain micronutrients, B, Ca, Mo, and Se (16, 785, 3.69, and 0.34 μg/g), were in A. strigosa. There is also evidence that the element content can be affected by the adopted cultivation procedure; however, the effects of the growing procedure can be significantly different when different species are considered. T. monococcum, grown by an organic procedure, presented lower concentrations of the major nutrients, while it demonstrated a modest increase in the micronutrients in the T. durum variety organic S. Cappelli, and the production procedure did not affect the elemental composition of the T. aestivum variety Verna. The survey also highlights that the studied species and the growing procedure affected the capacity to accumulate and translocate trace hazardous elements for human health at the seed level.
... Most developing countries, especially arid and semi-arid regions of the world, are facing water scarcity problems that directly affect the performance of different crops. Several studies have been carried out on foxtail millet, little millet, finger millet, proso millet, and wild millet and reported a significant decrease in yield performance when grown under drought-prone conditions (Satyavathi et al., 2019;Numan et al., 2021). Likewise, up to 80-90% yield loss was recorded in studied finger millet subjected to drought after 3-4 weeks of sowing (Maqsood and Ali, 2007). ...
... Similarly, India, Japan, China, France, and some East African countries have conserved the majority of foxtail millet germplasm. Correspondingly, barnyard millet is in India and Japan; proso millet is in India, China, Russia, and Ukraine; Kodo millet is in the USA and USA; and the major collections of little millet germplasm is conserved in India (Numan et al., 2021). Institute-wise, the largest collection center for small millets conserved across the countries are little millet (1253), Kodo millet (2180), and finger millet (9522) at NBPGR, India; foxtail millet (26,233) at ICS-CAAS, China; barnyard millet (3671) at NIAS, Japan; proso millet (8778) at NRSRIPI, Russia. ...
... Only a limited genomic infrastructure (genetic markers and mapping databases) is available for tef (Tadele, 2019). Hence, advanced genome-based marker-assisted breeding of tef has remained mostly out of reach (Bekana & Assefa, 2021;Numan et al., 2021). To the best of our knowledge, this is the first GWAS of tef under contrasting water regimes. ...
Article
Full-text available
Tef [Eragrostis tef (Zucc.) Trotter] is an allotetraploid (2n = 4x = 40) C4 cereal crop, endemic to Ethiopia and mainly cultivated in the Horn of Africa. Tef is characterized by high grain and feed nutritional qualities, and resilience to abiotic and biotic stresses, thus it holds a great potential to sustain food and nutrition security in Africa and other parts of the world. The objective of this study was to identify genomic regions associated with responses to contrasting water regimes, as a basis for future improvement. A tef diversity panel was genotyped with 28,837 single nucleotide polymorphisms (SNPs) and phenotyped for productivity, lodging, and morpho-physiological traits along two seasons (2020 and 2021) under well-watered and water-limited treatments. A genome-wide association study was performed to identify genomic regions associated with key traits for tef breeding. A total of 107 SNPs were associated with one or more of the studied traits, resulting in 138 marker–trait associations (MTAs) detected under both water treatments. Of these, 22 SNPs were associated with more than one MTA, showing either multiple trait (pleiotropic) or multiple environment associations, or both. A particularly strong association was found between grain yield, lodging, and time to heading. These findings open new avenues to further research on the genetic basis and physiological mechanisms underlying major traits in tef, as well as to marker-assisted breeding of drought-resilient tef cultivars.
... There were efforts to improve the productivity of tef through the use of nitrogen and phosphorus fertilizers, gene editing [1,18], and other agronomic practices like tillage [19], seed rate, and method of planting [20] under the rainfed production system. However, the applicability of any technology varies significantly over the location, season, crop, variety, and management and production systems; thus, the majority of the farmers were forced to use blanket recommendations made at the national level for the rainfed system. ...
Article
Full-text available
Lodging, poor crop varieties and nitrogen management are among the main tef cultivation problems in acidic soils of northwestern Ethiopia. Though Si has been shown to improve crop yield and lodging resistance, knowledge of its effect on tef, along genotypes and nitrogen, is yet to be uncovered. Therefore, a 4 × 2 × 2 factorial field experiment was conducted on fixed experimental plot at the Koga irrigation scheme to assess yield and lodging responses of tef varieties to nitrogen and silicon fertilizer rates during two consecutive years of 2021 and 2022. The experiment comprised four nitrogen levels: 0 (N1), 23 (N2), 46 (N3), and 92 kg N ha⁻¹(N4), two Si levels: 0 (Si1) and 485 (Si2) kg ha⁻¹, and two improved varieties: Hiber-1 (V1) and Quncho (V2) treatment combinations, which were replicated four times. Results showed that regardless of silicon supply and variety, nitrogen had a significant effect (p < .0001) on agronomic attributes of tef grain yield, biomass yield, harvest index, chlorophyll content, plant height, panicle length, leaf area index, and the number of plants m⁻² over the two years. Application of N4, N3, and N2 improved grain yield by 166.9, 126.2, and 75.2 % over N1, respectively. The harvest index showed a declining trend with nitrogen rates, which ranged from 36.1 to 26.5 %. Hiber-1 showed a significantly (p < .01) higher panicle length than Quncho. The interaction of nitrogen, silicon, and variety significantly (p < .001) affected lodging index, with a minimum lodging index of 0 % from V1Si1N1 and a maximum lodging index (71.9 %) from V2Si1N4. Maximum net return (2552.6 USD) was obtained from V1Si1N4, while the marginal rate of return (6961.7 %) from V1Si1N3. Therefore, it can be concluded that genotype and optimum nitrogen can maximize yield and lodging resistance of tef, while silicon in the form of carbonized rice husk results no significant effect on tef lodging.
... As a C 4 crop tef is adapted to grow under adverse environmental conditions such as imposed by drought, salinity, and waterlogging and at elevations ranging from 1800 and 2200 m above sea level (Ereful et al. 2022;Girija et al. 2022a). Research into tef is limited (Numan et al. 2021), which is compromising it achieving its potential. For example, despite its large area of cultivation, tef yields in Ethiopia remain far below as compared to wheat and maize (Cochrane and Bekele 2018). ...
Article
Full-text available
Main conclusion Salinity induced metabolite responses resulted in differential accumulation of flavonoids and antioxidant metabolites in shoots and roots suggesting improved antioxidant capacity in providing salt-adaptive phenotype of tef seedling. Abstract Tef [(Eragrostis tef) (Zucc.) Trotter] is an important ‘cash crop’ of Ethiopia grown mainly for human food, and development of elite tef cultivars with better performance is vital to Ethiopian farmers and breeders. Soil salinity is one of the key constraints that affects tef yield in the Ethiopian lowlands and Rift valley where cultivation of tef is limited. Being a minor crop, the responses of tef towards salinity is unknown. Salinity involves physiological and metabolite reprogramming that can have major impact on germination and seedling establishment. Here we evaluate the in vitro effect of NaCl on tef germination and associate this with metabolomic approaches to suggest salt tolerance mechanisms. In this study, 19 tef varieties were screened for NaCl tolerance and were investigated using untargeted metabolomics. Screened tef varieties showed differential germination rates with NaCl treatment varying from < 20 to 100%. Viable seedlings exposed to NaCl exhibited purple-red pigment accumulation in the roots except for Beten and Tullu nasy varieties. Metabolite comparisons between shoots and roots showed significant differences and, in particular, roots of salt tolerant tef varieties accumulated flavonoid derivatives as well as sugars and cell wall associated metabolites. These metabolic changes were correlated with patterns of antioxidant capacities and total flavonoid content in shoots and roots and suggested a mitigating response by tef to salinity. Our study highlights the role of flavonoid accumulation following salt stress on tef seedlings and further these findings could be used as targets for selective tef breeding.
... In tef, lodging is a very common devastating phenomenon, and hence, it is a major target for tef breeding (Assefa et al., 2011;Numan et al., 2021;Zeid et al., 2010). A collection of $220 tef lines tested under four environments demonstrated a wide genetic diversity in lodging, coupled with an intermediate level of heritability estimates (Ben-Zeev, 2022). ...
Article
Full-text available
Societal Impact Statement Crop diversification is considered key to ensuring agricultural sustainability and food security. Tef ( Eragrostis tef (Zucc.) Trotter) is a cereal crop grown mainly in Ethiopia, where it thrives in a wide range of environments, including stress‐prone habitats. It is considered a promising new crop and is gaining popularity in cultivation and in research worldwide. Lodging, the greatest factor limiting tef productivity and its wide adoption, was targeted in this study. The results highlight the central role of root traits in tef lodging, thus paving the way to reducing lodging and improving tef productivity, crop diversification, and food security. Summary Lodging is the most prominent yield‐restricting problem in tef ( Eragrostis tef (Zucc.) Trotter) cultivation, responsible for 30%–50% yield loss. A significant advance in lodging resistance has been achieved in various cereals by reducing plant height. In this study, we investigated the role of crown root morphology and anatomy, rather than plant height, in tef lodging. Twenty‐eight tef lines, representing a wide diversity in lodging tendencies and major phenotypic traits, were tested under two field environments to investigate tef lodging and related traits. Four selected lines were subjected to more intense sampling as well as anatomical analysis of crown roots. In both field studies, taller lines were associated with greater lodging shortly before flowering, but not at plant maturity, whereas crown root diameter exhibited an association with reduced lodging, which became highly significant at maturity. Moreover, a greater proportion of root cortical aerenchyma developed in lodging‐susceptible genotypes, possibly reducing plant anchorage. A positive association between grain yield and lodging presents a major challenge for tef breeding. Our results suggest that root traits play a central role in tef lodging responses. We propose that semi‐dwarfism should be complemented by targeting root traits, to promote lodging resistance in tef.
Article
Full-text available
Traditional plant breeding has helped to increase food production dramatically over the past five decades, and many countries have managed to produce enough food for the growing population, particularly in the developing world [...]
Chapter
Full-text available
The term “millet” refers to a varied group of small-seeded annual C4 panicoid grasses, the biomass of which is used as feed and the seeds as food. A vital climate-resilient nutrimillet with a wealth of elite genes and alleles is finger millet. The most effective and long-term tactics for increasing abiotic stress resistance in millet crops could be found in advanced biotechnology applications, like “omics” approaches. Abiotic stress tolerance-boosting strategies for millet crops could be discovered in cutting-edge biotechnology applications like “omics” techniques. An enhanced breeding method called genomics-assisted breeding takes into account both phenotypic selection and genetic information at the same time when creating phenotypes also can contribute to the abiotic stress tolerant variety development of the specie. In addition, application of novel technologies viz. gene manipulation technologies, tissue culture techniques, genetic diversity assessments, and germplasm conservation techniques can contribute to the enhancement of abiotic stress tolerance capacity of finger millet. In this chapter is discussing the various research approaches that have been focused to improve abiotic stress tolerance in finger millet. Also, this chapter provides the specifics of the phenomic and genomic methods used to improve finger millet.
Article
Full-text available
Crop improvement programmes began with traditional breeding practices since the inception of agriculture. Farmers and plant breeders continue to use these strategies for crop improvement due to their broad application in modifying crop genetic compositions. Nonetheless, conventional breeding has significant downsides in regard to effort and time. Crop productivity seems to be hitting a plateau as a consequence of environmental issues and the scarcity of agricultural land. Therefore, continuous pursuit of advancement in crop improvement is essential. Recent technical innovations have resulted in a revolutionary shift in the pattern of breeding methods, leaning further towards molecular approaches. Among the promising approaches, marker-assisted selection, QTL mapping, omics-assisted breeding, genome-wide association studies and genome editing have lately gained prominence. Several governments have progressively relaxed their restrictions relating to genome editing. The present review highlights the evolutionary and revolutionary approaches that have been utilized for crop improvement in a bid to produce climate-resilient crops observing the consequence of climate change. Additionally, it will contribute to the comprehension of plant breeding succession so far. Investing in advanced sequencing technologies and bioinformatics will deepen our understanding of genetic variations and their functional implications, contributing to breakthroughs in crop improvement and biodiversity conservation.
Article
Full-text available
Silicon (Si) is one of the beneficial plant mineral nutrients which is known to improve biotic and abiotic stress resilience and productivity in several crops. However, its beneficial role in underutilized or “orphan” crop such as tef [Eragrostis tef (Zucc.) Trotter] has never been studied before. In this study, we investigated the effect of Si application on tef plant performance. Plants were grown in soil with or without exogenous application of Na2SiO3 (0, 1.0, 2.0, 3.0, 4.0, and 5.0 mM), and biomass and grain yield, mineral content, chlorophyll content, plant height, and expression patterns of putative Si transporter genes were studied. Silicon application significantly increased grain yield (100%) at 3.0 mM Si, and aboveground biomass yield by 45% at 5.0 mM Si, while it had no effect on plant height. The observed increase in grain yield appears to be due to enhanced stress resilience and increased total chlorophyll content. Increasing the level of Si increased shoot Si and Na content while it significantly decreased the content of other minerals including K, Ca, Mg, P, S, Fe, and Mn in the shoot, which is likely due to the use of Na containing Si amendment. A slight decrease in grain Ca, P, S, and Mn was also observed with increasing Si treatment. The increase in Si content with increasing Si levels prompted us to analyze the expression of Si transporter genes. The tef genome contains seven putative Si transporters which showed high homology with influx and efflux Lsi transporters reported in various plant species including rice. The tef Lsi homologs were deferentially expressed between tissues (roots, leaves, nodes, and inflorescences) and in response to Si, suggesting that they may play a role in Si uptake and/or translocation. Taken together, these results show that Si application improves stress resilience and yield and regulates the expression of putative Si transporter genes. However, further study is needed to determine the physiological function of the putative Si transporters, and to study the effect of field application of Si on tef productivity.
Article
Full-text available
Tillering is an important biomass yield component trait in switchgrass (Panicum virgatum L.). Teosinte branched 1 (tb1)/Branched 1 (BRC1) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1a-Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.
Article
Full-text available
The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4–GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4–GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4–GIF1 with CRISPR–Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF–GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.
Article
Full-text available
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.
Article
Full-text available
Teff is a dominantly cultivated and stable crop in Ethiopia primarily grown for its grain which is used for preparing injera. In spite of its importance, the productivity is very low due to many factors among them, and poor agronomic practices are the major ones. In view of this, a field experiment, under rain-fed condition, was conducted at Laelay Machew district with the objective of evaluating the response of teff to seeding rate and methods of sowing during 2017/18 main cropping season. The experiment comprised four levels of seeding rate (10, 15, 20, and 25 kg/ha) and two methods of sowing (broad casting and row planting), and the experiment was laid in a 2 × 4 factorial arrangement in randomized complete block design (RCBD), replicated three times. Data related to phenology, growth, yield, and yield attributes were collected and analyzed using SAS software. Results indicated that days to panicle emergence, plant height, total number of tillers, productive tillers, main panicle seed weight, thousand-seed weight, panicle length, and harvest index were significantly (P
Article
Full-text available
Teff is an important food crop that serves to prepare Injera-flat-bread. It is cultivated worldwide and is particularly susceptible to lodging. A diverse collection of teff [Eragrostis tef (Zucc.) Trotter] populations was characterized for a wide range of traits, ranging from agronomic to final Injera sensory parameters, under well-irrigated Mediterranean spring conditions. The populations tested were collected from single plants presenting lodging resistance at the site of collection and their traits were characterized herein. An early type of lodging was observed, which was most likely triggered by a fast and sharp inflorescence weight increase. Other populations were ‘strong’ enough to carry the inflorescence during most of the grain-filling period, up to a point where strong lodging occurred and plants where totally bent to the ground. Three mixed color seed populations were established from a single plant. These were separated into ‘white’ and ‘brown’ seeds and were characterized separately under field conditions. The newly ‘brown’ populations appear to be the result of a rather recent non-self (external) airborne fertilization from a dark pollen donor. Some of these hybrids were found to be promising in terms of Injera sensory traits. The population of these studies might serve as breeding material. Integration between a wide range of parameters and the correlations obtained between agronomic and sensory traits might improve our ability to breed towards a “real world” better end-product.
Preprint
Full-text available
Tillering is an important biomass yield component trait in switchgrass ( Panicum virgatum L .). Teosinte branched 1 ( tb1 )/ Branched 1 ( BRC1 ) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained non-chimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller production in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free T1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1 a- Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass. Highlight Solid non-chimeric mutants were successfully isolated from CRISPR/Cas9-induced chimeric mutants using nodal culture. Teosinte branched 1 ( tb1 ) genes are involved in various pathways to regulate tillering in switchgrass.
Article
Full-text available
Cpf1, an endonuclease of the class 2 CRISPR family, fills the gaps that were previously faced in the world of genome engineering tools, which include the TALEN, ZFN, and CRISPR/Cas9. Other simultaneously discovered nucleases were not able to carry out re-engineering at the same region due to the loss of a target site after first-time engineering. Cpf1 acts as a dual nuclease, functioning as an endoribonuclease to process crRNA and endodeoxyribonuclease to cleave target sequences and generate double-stranded breaks. Additionally, Cpf1 allows for multiplexed genome editing, as a single crRNA array transcript can target multiple loci in the genome. The CRISPR/Cpf1 system enables gene deletion, insertion, base editing, and locus tagging in monocot as well as in dicot plants with fewer off-target effects. This tool has been efficiently demonstrated into tobacco, rice, soybean, wheat, etc. This review covers the development and applications of Cpf1 mediated genome editing technology in plants.
Preprint
Background CRISPR/Cas9-based genome editing holds great promise to accelerate the development of new crop varieties by providing a powerful tool to modify the genomic regions controlling major agronomic traits. To diversify the set of tools available for wheat genome engineering, we have established a tRNA-based multiplex gene editing strategy for hexaploid wheat. Results The functionality of the various CRISPR/Cas9 components was assessed using the transient expression in the wheat protoplasts followed by next-generation sequencing (NGS) of the targeted genomic regions. The efficiency of wheat codon-optimized Cas9 for targeted gene editing in wheat was validated. Multiple single guide RNAs (gRNAs) were evaluated for the ability to edit the homoeologous copies of four genes affecting some important agronomic traits in wheat. Low correspondence was found between the gRNA efficiency predicted bioinformatically and that assessed in the transient expression assay. A multiplex gene editing construct with several gRNA-tRNA units under the control of a single promoter for the RNA polymerase III generated indels at the targets sites with the efficiency comparable to that obtained for a single gRNA construct. Conclusions By integrating the protoplast transformation assay with multiplexed NGS, it is possible to perform fast functional screens for a large number of gRNAs and to optimize constructs for effective editing of multiple independent targets in the wheat genome. The multiplexing capacity of the tandemly arrayed tRNA–gRNA construct is well suited for the simultaneous editing of the redundant gene copies in the allopolyploid genomes or genomic regions beneficially affecting multiple agronomic traits. A polycistronic gene construct that can be quickly assembled using the Golden Gate reaction along with the wheat codon optimized Cas9 will further expand the set of tools available for engineering the wheat genome.